Targeted neoepitope vectors and methods therefor

ABSTRACT

Systems and methods are presented that allow for selection of tumor neoepitopes that are then used to generate recombinant nucleic acids that encode one or more polytopes that are optimized for proper trafficking and processing. In preferred methods, the polytopes are encoded in a plasmid and/or a viral expression system for use as a therapeutic agent.

This application claims priority to our copending U.S. provisionalpatent application with the Ser. No. 62/489,102, filed Apr. 24, 2017.

FIELD OF THE INVENTION

The field of the invention is compositions and methods of improvedneoepitope-based immune therapeutics, especially as it relates torecombinant nucleic acid therapeutics in the treatment of cancer.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Cancer immunotherapies targeting certain antigens common to a specificcancer have led to remarkable responses in some patients. Unfortunately,many patients failed to respond to such immunotherapy despite apparentexpression of the same antigen. One possible reason for such failurecould be that various effector cells of the immune system may not havebeen present in sufficient quantities, or may have been exhausted.Moreover, intracellular antigen processing and HLA variability amongpatients may have led to insufficient processing of the antigen and/orantigen display, leading to a therapeutically ineffective or lackingresponse.

To increase the selection of targets for immune therapy, randommutations have more recently been considered since some random mutationsin tumor cells may give rise to unique tumor specific antigens(neoepitopes). As such, and at least conceptually, neoepitopes mayprovide a unique precision target for immunotherapy. Additionally, ithas been shown that cytolytic T-cell responses can be triggered by verysmall quantities of peptides (e.g., Sykulev et al., Immunity, Volume 4,Issue 6, p 565-571, 1 Jun. 1996). Moreover, due to the relatively largenumber of mutations in many cancers, the number of possible targets isrelatively high. In view of these findings, the identification of cancerneoepitopes as therapeutic targets has attracted much attention.Unfortunately, current data appear to suggest that all or almost allcancer neoepitopes are unique to a patient and specific tumor and failto provide any specific indication as to which neoepitope may be usefulfor an immunotherapeutic agent that is therapeutically effective.

To overcome at least some of the problems associated with large numbersof possible targets for immune therapy, the neoepitopes can be filteredfor the type of mutation (e.g., to ascertain missense or nonsensemutation), the level of transcription to confirm transcription of themutated gene, and to confirm protein expression. Moreover, the sofiltered neoepitope may be further analyzed for specific binding to thepatient's HLA system as described in WO 2016/172722. While such systemadvantageously reduces the relatively large number of potentialneoepitopes, the significance of these neoepitopes with respect totreatment outcome remains uncertain. Still further, and especially wheremultiple peptides are to be expressed in an antigen presenting cell(e.g., dendritic cell), processing of precursor proteins to generate theneoepitopes is not fully understood and contributes as such to the lackof predictability of therapeutic success.

Immune therapy can be performed using at least two conceptually distinctapproaches, with the first approach based on DNA vaccination and thesecond approach based on use of a recombinant virus that encodes one ormore antigens that are expressed in a cell infected with the virus. Forexample, clinical trials have suggested that plasmid DNA vaccines aresafe and immunologically effective in humans at doses of 300 mcg ofplasmid DNA encoding HIV rev and env proteins when administeredintramuscularly. Such DNA vaccination elicited antigen-specific, IFNgamma-secreting T cell responses in HIV-seronegative patients (J.Infect. Dis. (2000) 181:476-83). In addition, results of a clinicaltrial targeting PSMA (prostate-specific membrane antigen) in patientswith prostate cancer using intradermal injections of plasmid DNA andadenovirus have been reported (see Eur. Urol. (2000), 38:208 217). Here,26 patients were immunized either in a prime/boost strategy with anadenoviral vector expressing PSMA followed by immunization with plasmidDNA encoding PSMA, or with plasmid DNA alone, and no significanttoxicity were observed. However, therapeutic efficacy of suchvaccinations, particularly in treatment of cancer has not beendemonstrated using such approaches. In still other examples, adenoviralexpression of cancer neoepitopes has been reported as described in US2017/0312351. While such approaches are highly specific towards thepatient and tumor of the patient, generation of sufficient quantities ofviral particles that encode one or more neoepitopes is time consuming.For example, virus production to generate a single dose of 10¹¹ virusparticles will often require 6-8 weeks and in some cases even longer,and multiple administrations are often required to elicit a therapeuticeffect. Depending on the type of cancer and growth speed, suchproduction time frame can be prohibitive. In addition, immunestimulation with virally expressed proteins only is often lesseffective, and additional treatment modalities such as cytokines arefrequently required to elicit a desirable therapeutic effect.

Thus, even though multiple methods of identification and delivery ofneoepitopes to various cells are known in the art, all or almost all ofthem suffer from various disadvantages, particularly in terms ofefficacy and time requirements. Consequently, it would be desirable tohave improved systems and methods for neoepitope selection andproduction that increases the likelihood of a therapeutic response inimmune therapy in an expedient fashion.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to various immune therapeuticcompositions and methods, and especially recombinant expression systemsin which multiple neoepitopes are combined to form a rational-designedpolypeptide with a trafficking signal to increase antigen processing andpresentation and to so enhance therapeutic efficacy. Additionally, thesystems and methods contemplated herein take advantage of multiple anddistinct vaccination modalities that will provide both, significantlyshortened time-to-first-vaccination periods and different andcomplementary modes of immune stimulation.

For example, where the first vaccination modality comprises a DNAvaccination that encodes a polytope (typically comprising multipleneoepitopes and/or TAAs), the vaccine can be prepared within a few daysand will provide a TLR stimulus (e.g., TLR9 stimulus), while the secondvaccination modality may comprise a recombinant bacterial or yeastvaccine that encodes the polytope (typically the same polytope as thefirst) and will so provide a different TRL stimulus (e.g., TRL1, TLR2,TLR5, etc.). In yet another example, the first vaccination modality maycomprise a bacterial or yeast vaccine that encodes the polytope and thesecond vaccination modality may comprise a recombinant virus thatencodes the polytope, which once again will provide distinct (andtypically complementary or even synergistic) innate immune stimuli.

As will be readily apparent, such multi-modality strategy willsubstantially reduce the time to generate the first vaccination as DNA,bacterial, and yeast vaccines can be prepared within a few days ratherthan several weeks as is the case with most viral vaccines. Moreover,due to the distinct forms of delivery, contemplated vaccine compositionsand methods will also take advantage of the distinct immune stimulatoryeffects provided by the different modalities and will as such beparticularly useful in a prime/boost regimen. Additionally, it should berecognized that contemplated systems and methods take advantage ofsubstantially the same polytope across the different vaccine modalities.In other words, once antigens with potential therapeutic effect aredetermined for a patient, a recombinant nucleic acid encoding theseantigens can be assembled into a polytope cassette that can then be usedacross multiple vaccine platforms.

In one aspect of the inventive subject matter, the inventors contemplatea method of generating recombinant expression constructs for use inimmune therapy in a mammal. Such methods will typically include a stepof generating a first recombinant nucleic acid having a sequence thatencodes a polytope, wherein the polytope comprises a plurality offiltered neoepitope sequences, wherein the polytope further comprises atrafficking element that directs the polytope to a sub-cellular locationselected from the group consisting of a recycling endosome, a sortingendosome, and a lysosome, and wherein the first recombinant nucleic acidcomprises a first promoter operably linked to the sequence that encodesthe polytope to drive expression of the polytope in the mammal. Inanother step, a second recombinant nucleic acid is generated having thesame sequence that encodes the polytope, wherein the second recombinantnucleic acid comprises a second promoter operably linked to the sequencethat encodes the polytope to drive expression of the polytope in anon-mammalian cell;

For example, in exemplary embodiments the first promoter may be aconstitutive promoter or a promoter that is inducible by hypoxia,IFN-gamma, or IL-8. Additionally, the trafficking element may be a CD1bleader sequence, a CD1a tail, a CD1c tail, or a LAMP1-transmembranesequence. Most typically, the filtered neoepitope sequences are filteredby comparing tumor versus matched normal of the same patient, and/orfiltered to have binding affinity to an MHC complex of equal or lessthan 200 nM. While in some aspects, the filtered neoepitope sequencesare calculated to bind to MHC-I and the trafficking element directs thepolytope to the recycling endosome, sorting endosome, or lysosome, inother aspects the filtered neoepitope sequences are calculated to bindto MHC-II and the trafficking element directs the polytope to therecycling endosome, sorting endosome, or lysosome.

Where desired, the first recombinant nucleic acid further may furthercomprise an additional sequence that encodes a second polytope, whereinthe second polytope comprises a second trafficking element that directsthe second polytope to a different sub-cellular location and wherein thesecond polytope comprises a second plurality of filtered neoepitopesequences. In some embodiments, at least one of the filtered neoepitopesequences and at least one of the second filtered neoepitope sequencesmay be the same.

It is still further contemplated that the first recombinant nucleic acidfurther comprises a sequence that encodes at least one of aco-stimulatory molecule, an immune stimulatory cytokine, and a proteinthat interferes with or down-regulates checkpoint inhibition. Forexample, suitable co-stimulatory molecules include CD80, CD86, CD30,CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L,TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and/or LFA3, while suitableimmune stimulatory cytokine include IL-2, IL-12, IL-15, IL-15 superagonist (ALT803), IL-21, IPS1, and/or LMP1, and/or suitable proteinsthat interfere include antibodies against or antagonists of CTLA-4,PD-1, TIM1 receptor, 2B4, and/or CD160.

While not limiting to the inventive subject matter, the firstrecombinant nucleic acid may be replicated in a bacterial cell or ayeast cell, and/or the first recombinant nucleic acid may be anexpression vector or a shuttle vector for generation of a recombinantvirus (e.g., adenovirus, optionally with at least one of an E1 and anE2b gene deleted). It is also contemplated that such methods may alsoinclude a step of formulating the first recombinant nucleic acid into apharmaceutical formulation for injection.

Most typically, the second promoter is a constitutive bacterial or ayeast promoter. Therefore, suitable non-mammalian cells include E. colicell and Saccharomyces cerevisiae. In such cases, methods may includethe additional steps of transfecting the second recombinant nucleic acidinto a bacterial cell or a yeast cell; expressing the polytope in thebacterial cell or the yeast cell; and formulating the bacterial cell orthe yeast cell into a pharmaceutical formulation for injection.

Consequently, the inventors also contemplate a recombinant bacterial oryeast expression vector for immune therapy of a mammal. Most preferably,such vector will include a recombinant nucleic acid having a sequencethat encodes a polytope operably linked to a bacterial or yeast promoterto drive expression of the polytope, wherein the polytope comprises atrafficking element that directs the polytope to a sub-cellular locationof a mammalian immune competent cell selected from the group consistingof recycling endosome, sorting endosome, and lysosome; and wherein thepolytope comprises a plurality of filtered neoepitope sequences.

Preferably, but not necessarily, the promoter is a constitutivepromoter, while the trafficking element is selected from the groupconsisting of a CD1b leader sequence, a CD1a tail, a CD1c tail, and aLAMP1-transmembrane sequence. As noted earlier, the filtered neoepitopesequences may be filtered by comparing tumor versus matched normal ofthe same patient, and the filtered neoepitope sequences bind to MHC-Iand/or MHC-II, and the trafficking element directs the polytope to therecycling endosome, sorting endosome, or lysosome. It is still furthercontemplated that the recombinant nucleic acid may also comprise anadditional sequence that encodes a second polytope, wherein the secondpolytope comprises a second trafficking element that directs the secondpolytope to a different sub-cellular location and wherein the secondpolytope comprises a second plurality of filtered neoepitope sequences.As before, at least one of the filtered neoepitope sequences and atleast one of the second filtered neoepitope sequences may be identical.

In still further contemplated aspects, the expression vector is abacterial expression vector or a yeast expression vector. Therefore,recombinant yeast cells and bacterial cells transfected with the vectorcontemplated above are particularly contemplated. These cells may thenbe formulated into a pharmaceutical composition comprising therecombinant yeast cells or bacterial cells.

In a further aspect of the on inventive subject matter, the inventorsalso contemplate a method of preparing first and second treatmentcompositions for an individual having a tumor. Such methods willtypically include a step of identifying a plurality of expressedneoepitope sequences from omics data of the tumor, wherein each of theexpressed neoepitope sequences have a calculated binding affinity ofequal or less than 500 nM to at least one of MHC-I and MHC-II of theindividual, and a further step of generating a first recombinant nucleicacid having a sequence that encodes a polytope, wherein the polytopecomprises the plurality of expressed neoepitope sequences. Preferably,the polytope further comprises a trafficking element that directs thepolytope to a sub-cellular location selected from the group consistingof a recycling endosome, a sorting endosome, and a lysosome, and thefirst recombinant nucleic acid comprises a first promoter operablylinked to the sequence that encodes the polytope to drive expression ofthe polytope in a cell of the individual. In another step, the firstrecombinant nucleic is formulated into a DNA vaccine formulation to soobtain the first treatment composition. In yet another step, a secondrecombinant nucleic acid is generated that includes the sequence thatencodes the polytope, wherein the second recombinant nucleic acidcomprises a second promoter operably linked to the sequence that encodesthe polytope to drive expression of the polytope in a bacterial cell ora yeast cell, and in a further step, the bacterial cell or the yeastcell is transfected with the second recombinant nucleic acid andexpressing the polytope in the bacterial cell or the yeast cell. In astill further step, the transfected bacterial cell or the yeast cell isformulated into a cell-based vaccine formulation to so obtain the secondtreatment composition.

Most typically, the expressed neoepitope sequences are identified usingincremental synchronous alignment of omics data from the tumor and omicsdata from a non-tumor sample of the same individual. It is furthergenerally preferred that the first recombinant nucleic acid is anexpression vector, and/or that the trafficking element is a CD1b leadersequence, a CD1a tail, a CD1c tail, or a LAMP1-transmembrane sequence.The second promoter is preferably a constitutive bacterial or a yeastpromoter, and the bacterial cell or the yeast cell is preferably E. colicell or Saccharomyces cerevisiae. Most typically, the cell-based vaccineformulation is formulated for injection. Furthermore, where desired,such method may further comprise a step of generating a thirdrecombinant nucleic acid that is a viral expression vector that includesthe sequence that encodes the polytope, wherein the third recombinantnucleic acid comprises a third promoter operably linked to the sequencethat encodes the polytope to drive expression of the polytope in a cellof the individual.

In still another aspect of the on inventive subject matter, theinventors also contemplate a method of preparing first and secondtreatment compositions for an individual having a tumor that includes astep of identifying a plurality of expressed neoepitope sequences fromomics data of the tumor, wherein the expressed neoepitope sequences havea calculated binding affinity of equal or less than 500 nM to at leastone of MHC-I and MHC-II of the individual; and a further step ofgenerating a first recombinant nucleic acid having a sequence thatencodes a polytope, wherein the polytope comprises the plurality ofexpressed neoepitope sequences, wherein the first recombinant nucleicacid is a viral expression vector. Preferably, the polytope furthercomprises a trafficking element that directs the polytope to asub-cellular location selected from the group consisting of a recyclingendosome, a sorting endosome, and a lysosome, and the first recombinantnucleic acid comprises a first promoter operably linked to the sequencethat encodes the polytope to drive expression of the polytope in a cellof the individual. In a further step, viral particles are generated fromthe viral expression vector and the viral particles are formulated intoa viral vaccine formulation to so obtain the first treatmentcomposition. Such methods will also include a step of generating asecond recombinant nucleic acid having the sequence that encodes thepolytope, wherein the second recombinant nucleic acid comprises a secondpromoter operably linked to the sequence that encodes the polytope todrive expression of the polytope in a non-mammalian cell, and a furtherstep of transfecting a bacterial cell or a yeast cell with the secondrecombinant nucleic acid and expressing the polytope in the bacterialcell or the yeast cell. The so transfected bacterial cell or the yeastcell is then formulated into a cell-based vaccine formulation to soobtain the second treatment composition.

Most typically, the plurality of expressed neoepitope sequences areidentified using incremental synchronous alignment of omics data fromthe tumor and omics data from a non-tumor sample of the same individual,and/or the trafficking element is a CD1b leader sequence, a CD1a tail, aCD1c tail, or a LAMP1-transmembrane sequence. Likewise, it is preferredthat the first promoter is a constitutive promoter or that the firstpromoter is inducible by hypoxia, IFN-gamma, or IL-8, and/or the secondpromoter is a constitutive bacterial or a yeast promoter. In furthercontemplated embodiments, the viral expression vector is an adenoviralexpression vector, optionally having E1 and E2b genes deleted. While notlimiting to the inventive subject matter, it is generally preferred thatthe non-mammalian cell or the yeast cell is an E. coli cell or aSaccharomyces cerevisiae cell, and/or that the viral vaccine formulationand the cell-based vaccine formulation are both formulated forinjection.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic representation of various neoepitope arrangements.

FIG. 2 is an exemplary and partial schematic for selecting preferredarrangements of neoepitopes.

Prior Art FIG. 3 is a schematic illustration of antigen processing inthe cytoplasm and MHC-I presentation.

Prior Art FIG. 4 is a schematic illustration of antigen processing inthe lysosomal and endosomal compartment and MHC-II presentation.

FIGS. 5A-5C are exemplary sequence arrangements for class I antigenprocessing in the cytoplasm and MHC-I presentation.

FIGS. 6A-6C are exemplary sequence arrangements for class I antigenprocessing in the cytoplasm and MHC-II presentation.

FIGS. 7A-7C are exemplary sequence arrangements for class II antigenprocessing in the cytoplasm and MHC-II presentation.

FIG. 8 is an exemplary prophylactic vaccination schedule for B16-F10melanoma.

FIGS. 9A-9C are graphs depicting exemplary results for the anti-tumorvaccination using subcutaneous injection of the vaccine.

FIGS. 10A-10C are graphs depicting exemplary results for the anti-tumorvaccination using intravenous injection of the vaccine.

FIGS. 11A-11E are graphs depicting exemplary results for selected DNAand viral vaccine compositions and selected routes of administration.

FIG. 12 is a graph depicting exemplary results for the anti-tumor effectusing DNA and viral vaccines with various compositions and routes.

DETAILED DESCRIPTION

The inventors have now discovered that various aspects in tumorantigen-based and/or neoepitope-based immune therapy can further beimproved by not only targeting the antigens or neoepitopes towardsspecific processing and cell surface presentation pathways, but also byusing different vaccine modalities that preferably trigger differentimmune stimulatory pathways.

Therefore, in addition to a viral cancer vaccine that is based on arecombinant virus that triggers in a host cell expression of tumorassociated or tumor specific antigens, the same (and/or additional)antigens may be expressed from a DNA vaccine and/or provided in yeastand/or bacterial cells that are genetically engineered to express theseantigens. For example, where a plasmid is used in a DNA vaccine, innateimmune response mechanisms against free DNA (e.g., TLR9- or STING-based)may be triggered along with the adaptive immune response based on invivo expression of the free DNA. In another example, where a viralexpression vector is employed as part of a viral vaccine in which avirus infects patient cells, such infection will typically triggerdifferent innate immune response mechanisms (typically TLR2, TLR4, TLR7, TLR 8, TLR 9). In still another example, where a bacterial or yeastvaccine is used in which the bacterium or yeast has expressed theantigen(s), such vaccine vaccination will once more trigger distinctinnate immune response mechanisms (typically TLR1-3 for bacterial andTLR1-4 for yeast). As will be readily appreciated, triggering of variousand distinct innate immune response mechanisms may provide complementaryor even synergistic enhancement of the vaccine compositions.

Thus, it should be appreciated that the compositions and methodspresented herein will preferably include use of at least two differentvaccine modalities. For example, the first modality may be a modalityselected from the group consisting of a DNA vaccine, protein vaccine, abacterial vaccine, a yeast vaccine, and a viral vaccine, while thesecond/subsequent modality may be another, different, modality selectedfrom the same group. Most preferably, the antigens present in any of themodalities will overlap or be the same to take advantage of aprime/boost effect upon repeated antigenic challenge.

In addition, it should be recognized that beyond the benefit oftriggering multiple distinct innate immune pathways, contemplatedcompositions and methods also allow for a rapid start of treatment if apatient with respect to the point in time at which the tumor relevantantigens in the patient were identified. Indeed, it should be recognizedthat a DNA vaccine can be prepared based on the relevant antigens withina few days, typically within less than a week and even less than 4 days,or even less 48 hours. Moreover, bacterial or yeast vaccines can be alsoprepared using (the same) antigens within a few days, typically withinless than 2 weeks, and more typically within less than 1 week. At thesame time, a viral vaccine can be prepared when the patient alreadyreceived the first vaccines (e.g., DNA vaccine, bacterial vaccine,and/or yeast vaccine).

Viewed from a different perspective, it should be appreciated that thecompositions and methods presented herein will include one or more tumorassociated antigens, tumor specific antigens, and/or neoepitopes thatare specific to the patient and the tumor in the patient to allow fortargeted treatment. Moreover, such treatment may advantageously betailored to achieve one or more specific immune reactions, including aninnate immune response, a CD4⁺ biased immune response, a CD8⁺ biasedimmune response, antibody biased immune response, and/or a stimulatedimmune response (e.g., reducing checkpoint inhibition and/or byactivation of immune competent cells using cytokines). Most typically,such effects are in achieved in the context of the neoepitopesoriginating from the recombinant nucleic acid that can be administeredvia one or more routes in one or more distinct formats (e.g., asrecombinant plasmid and as recombinant virus).

Antigens

With respect to suitable therapeutic antigens it is contemplated thatvarious antigens are deemed suitable for use herein. However,particularly preferred antigens include tumor associated antigens (e.g.,CEA, MUC1, brachyury), tumor specific antigens (e.g., HER2, PSA, PSMA,etc.), and especially tumor and patient specific antigens (i.e.,neoepitopes). Neoepitopes can be characterized as expressed randommutations in tumor cells that created unique and tumor specificantigens. Therefore, viewed from a different perspective, neoepitopesmay be identified by considering the type (e.g., deletion, insertion,transversion, transition, translocation) and impact of the mutation(e.g., non-sense, missense, frame shift, etc.), which may as such serveas a content filter through which silent and other non-relevant (e.g.,non-expressed) mutations are eliminated. It should also be appreciatedthat neoepitope sequences can be defined as sequence stretches withrelatively short length (e.g., 8-12 mers or 14-20mers) wherein suchstretches will include the change(s) in the amino acid sequences. Mosttypically, but not necessarily, the changed amino acid will be at ornear the central amino acid position. For example, a typical neoepitopemay have the structure of A₄-N-A₄, or A₃-N-A₅, or A₂-N-A₇, or A₅-N-A₃,or A₇-N-A₂, where A is a proteinogenic wild type or normal (i.e., fromcorresponding healthy tissue of the same patient) amino acid and N is achanged amino acid (relative to wild type or relative to matchednormal). Therefore, the neoepitope sequences contemplated herein includesequence stretches with relatively short length (e.g., 5-30 mers, moretypically 8-12 mers, or 14-20 mers) wherein such stretches include thechange(s) in the amino acid sequences. Where desired, additional aminoacids may be placed upstream or downstream of the changed amino acid,for example, to allow for additional antigen processing in the variouscompartments (e.g., for proteasome processing in the cytosol, orspecific protease processing in the endosomal and/or lysosomalcompartments) of a cell.

Thus, it should be appreciated that a single amino acid change may bepresented in numerous neoepitope sequences that include the changedamino acid, depending on the position of the changed amino acid.Advantageously, such sequence variability allows for multiple choices ofneoepitopes and as such increases the number of potentially usefultargets that can then be selected on the basis of one or more desirabletraits (e.g., highest affinity to a patient HLA-type, highest structuralstability, etc.). Most typically, neoepitopes will be calculated to havea length of between 2-50 amino acids, more typically between 5-30 aminoacids, and most typically between 8-12 amino acids, or 14-20 aminoacids, with the changed amino acid preferably centrally located orotherwise situated in a manner that improves its binding to MHC. Forexample, where the epitope is to be presented by the MHC-I complex, atypical neoepitope length will be about 8-12 amino acids, while thetypical neoepitope length for presentation via MHC-II complex will havea length of about 14-20 amino acids. As will be readily appreciated,since the position of the changed amino acid in the neoepitope may beother than central, the actual peptide sequence and with that actualtopology of the neoepitope may vary considerably, and the neoepitopesequence with a desired binding affinity to the MHC-I or MHC-IIpresentation and/or desired protease processing will typically dictatethe particular sequence.

Of course, it should be appreciated that the identification or discoveryof neoepitopes may start with a variety of biological materials,including fresh biopsies, frozen, or otherwise preserved tissue or cellsamples, circulating tumor cells, exosomes, various body fluids (andespecially blood), etc. Therefore, suitable methods of omics analysisinclude nucleic acid sequencing, and particularly NGS methods operatingon DNA (e.g., Illumina sequencing, ion torrent sequencing, 454pyrosequencing, nanopore sequencing, etc.), RNA sequencing (e.g.,RNAseq, reverse transcription based sequencing, etc.), and in some casesprotein sequencing or mass spectroscopy based sequencing (e.g., SRM,MRM, CRM, etc.).

As such, and particularly for nucleic acid based sequencing, it shouldbe particularly recognized that high-throughput genome sequencing of atumor tissue will allow for rapid identification of neoepitopes.However, it must be appreciated that where the so obtained sequenceinformation is compared against a standard reference, the normallyoccurring inter-patient variation (e.g., due to SNPs, short indels,different number of repeats, etc.) as well as heterozygosity will resultin a relatively large number of potential false positive neoepitopes.Notably, such inaccuracies can be eliminated where a tumor sample of apatient is compared against a matched normal (i.e., non-tumor) sample ofthe same patient.

In one especially preferred aspect of the inventive subject matter, DNAanalysis is performed by whole genome sequencing and/or exome sequencing(typically at a coverage depth of at least 10×, more typically at least20×) of both tumor and matched normal sample. Alternatively, DNA datamay also be provided from an already established sequence record (e.g.,SAM, BAM, FASTA, FASTQ, or VCF file) from a prior sequence determinationof the same patient. Therefore, data sets suitable for use hereininclude unprocessed or processed data sets, and exemplary preferred datasets include those having BAM format, SAM format, GAR format, FASTQformat, or FASTA format, as well as BAMBAM, SAMBAM, and VCF data sets.However, it is especially preferred that the data sets are provided inBAM format or as BAMBAM diff objects as is described in US2012/0059670A1and US2012/0066001A1. Moreover, it should be noted that the data setsare reflective of a tumor and a matched normal sample of the samepatient. Thus, genetic germ line alterations not giving rise to thetumor (e.g., silent mutation, SNP, etc.) can be excluded. Of course, itshould be recognized that the tumor sample may be from an initial tumor,from the tumor upon start of treatment, from a recurrent tumor and/ormetastatic site, etc. In most cases, the matched normal sample of thepatient is blood, or a non-diseased tissue from the same tissue type asthe tumor.

Likewise, the computational analysis of the sequence data may beperformed in numerous manners. In most preferred methods, however,analysis is performed in silico by incremental location-guidedsynchronous alignment of tumor and normal samples as, for example,disclosed in US 2012/0059670 and US 2012/0066001 using BAM files and BAMservers. Such analysis advantageously reduces false positive neoepitopesand significantly reduces demands on memory and computational resources.

It should be noted that any language directed to a computer should beread to include any suitable combination of computing devices, includingservers, interfaces, systems, databases, agents, peers, engines,controllers, or other types of computing devices operating individuallyor collectively. One should appreciate the computing devices comprise aprocessor configured to execute software instructions stored on atangible, non-transitory computer readable storage medium (e.g., harddrive, solid state drive, RAM, flash, ROM, etc.). The softwareinstructions preferably configure the computing device to provide theroles, responsibilities, or other functionality as discussed below withrespect to the disclosed apparatus. Further, the disclosed technologiescan be embodied as a computer program product that includes anon-transitory computer readable medium storing the softwareinstructions that causes a processor to execute the disclosed stepsassociated with implementations of computer-based algorithms, processes,methods, or other instructions. In especially preferred embodiments, thevarious servers, systems, databases, or interfaces exchange data usingstandardized protocols or algorithms, possibly based on HTTP, HTTPS,AES, public-private key exchanges, web service APIs, known financialtransaction protocols, or other electronic information exchangingmethods. Data exchanges among devices can be conducted over apacket-switched network, the Internet, LAN, WAN, VPN, or other type ofpacket switched network; a circuit switched network; cell switchednetwork; or other type of network.

Viewed from a different perspective, a patient- and cancer-specific insilico collection of sequences can be established that encodeneoepitopes having a predetermined length of, for example, between 5 and25 amino acids and include at least one changed amino acid. Suchcollection will typically include for each changed amino acid at leasttwo, at least three, at least four, at least five, or at least sixmembers in which the position of the changed amino acid is notidentical. Such collection advantageously increases potential candidatemolecules suitable for immune therapy and can then be used for furtherfiltering (e.g., by sub-cellular location, transcription/expressionlevel, MHC-I and/or II affinity, etc.) as is described in more detailbelow. Of course, it should be appreciated that these neoepitopesequences can be readily back-translated into the corresponding nucleicacid sequences to so generate a nucleic acid sequence that encodes theneoepitope. Most typically, but not necessarily, such back-translationwill take into account the proper codon usage of the organism in whichthe nucleic acid is being expressed.

For example, and using synchronous location guided analysis to tumor andmatched normal sequence data, the inventors previously identifiedvarious cancer neoepitopes from a variety of cancers and patients,including the following cancer types: BLCA, BRCA, CESC, COAD, DLBC, GBM,HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PRAD, READ,SARC, SKCM, STAD, THCA, and UCEC. Exemplary neoepitope data for thesecancers can be found in International application WO 2016/172722,incorporated by reference herein.

Depending on the type and stage of the cancer, as well as the patient'simmune status it should be recognized that not all of the identifiedneoepitopes will necessarily lead to a therapeutically equally effectivereaction in a patient. Indeed, it is well known in the art that only afraction of neoepitopes will generate an immune response. To increaselikelihood of a therapeutically desirable response, the initiallyidentified neoepitopes can be further filtered. Of course, it should beappreciated that downstream analysis need not take into account silentmutations for the purpose of the methods presented herein. However,preferred mutation analyses will provide in addition to the particulartype of mutation (e.g., deletion, insertion, transversion, transition,translocation) also information of the impact of the mutation (e.g.,non-sense, missense, etc.) and may as such serve as a first contentfilter through which silent mutations are eliminated. For example,neoepitopes can be selected for further consideration where the mutationis a frame-shift, non-sense, and/or missense mutation.

In a further filtering approach, neoepitopes may also be subject todetailed analysis for sub-cellular location parameters. For example,neoepitope sequences may be selected for further consideration if theneoepitopes are identified as having a membrane associated location(e.g., are located at the outside of a cell membrane of a cell) and/orif an in silico structural calculation confirms that the neoepitope islikely to be solvent exposed, or presents a structurally stable epitope(e.g., J Exp Med 2014), etc.

With respect to filtering neoepitopes, it is generally contemplated thatneoepitopes are especially suitable for use herein where omics (orother) analysis reveals that the neoepitope is actually expressed.Identification of expression and expression level of a neoepitope can beperformed in all manners known in the art and preferred methods includequantitative RNA (hnRNA or mRNA) analysis and/or quantitative proteomicsanalysis. Most typically, the threshold level for inclusion ofneoepitopes will be an expression level of at least 20%, at least 30%,at least 40%, or at least 50% of expression level of the correspondingmatched normal sequence, thus ensuring that the (neo)epitope is at leastpotentially ‘visible’ to the immune system. Consequently, it isgenerally preferred that the omics analysis also includes an analysis ofgene expression (transcriptomic analysis) to so help identify the levelof expression for the gene with a mutation.

There are numerous methods of transcriptomic analysis known in the art,and all of the known methods are deemed suitable for use herein. Forexample, preferred materials include mRNA and primary transcripts(hnRNA), and RNA sequence information may be obtained from reversetranscribed polyA⁺-RNA, which is in turn obtained from a tumor sampleand a matched normal (healthy) sample of the same patient. Likewise, itshould be noted that while polyA⁺-RNA is typically preferred as arepresentation of the transcriptome, other forms of RNA (hn-RNA,non-polyadenylated RNA, siRNA, miRNA, etc.) are also deemed suitable foruse herein. Preferred methods include quantitative RNA (hnRNA or mRNA)analysis and/or quantitative proteomics analysis, especially includingRNAseq. In other aspects, RNA quantification and sequencing is performedusing RNAseq, qPCR and/or rtPCR based methods, although variousalternative methods (e.g., solid phase hybridization-based methods) arealso deemed suitable. Viewed from another perspective, transcriptomicanalysis may be suitable (alone or in combination with genomic analysis)to identify and quantify genes having a cancer- and patient-specificmutation.

Similarly, proteomics analysis can be performed in numerous manners toascertain actual translation of the RNA of the neoepitope, and all knownmanners of proteomics analysis are contemplated herein. However,particularly preferred proteomics methods include antibody-based methodsand mass spectroscopic methods. Moreover, it should be noted that theproteomics analysis may not only provide qualitative or quantitativeinformation about the protein per se, but may also include proteinactivity data where the protein has catalytic or other functionalactivity. One exemplary technique for conducting proteomic assays isdescribed in U.S. Pat. No. 7,473,532, incorporated by reference herein.Further suitable methods of identification and even quantification ofprotein expression include various mass spectroscopic analyses (e.g.,selective reaction monitoring (SRM), multiple reaction monitoring (MRM),and consecutive reaction monitoring (CRM)). Consequently, it should beappreciated that the above methods will provide patient and tumorspecific neoepitopes, which may be further filtered by sub-cellularlocation of the protein containing the neoepitope (e.g., membranelocation), the expression strength (e.g., overexpressed as compared tomatched normal of the same patient), etc.

In yet another aspect of filtering, the neoepitopes may be comparedagainst a database that contains known human sequences (e.g., of thepatient or a collection of patients) to so avoid use of ahuman-identical sequence. Moreover, filtering may also include removalof neoepitope sequences that are due to SNPs in the patient where theSNPs are present in both the tumor and the matched normal sequence. Forexample, dbSNP (The Single Nucleotide Polymorphism Database) is a freepublic archive for genetic variation within and across different speciesdeveloped and hosted by the National Center for BiotechnologyInformation (NCBI) in collaboration with the National Human GenomeResearch Institute (NHGRI). Although the name of the database implies acollection of one class of polymorphisms only (single nucleotidepolymorphisms (SNPs)), it in fact contains a relatively wide range ofmolecular variation: (1) SNPs, (2) short deletion and insertionpolymorphisms (indels/DIPs), (3) microsatellite markers or short tandemrepeats (STRs), (4) multinucleotide polymorphisms (MNPs), (5)heterozygous sequences, and (6) named variants. The dbSNP acceptsapparently neutral polymorphisms, polymorphisms corresponding to knownphenotypes, and regions of no variation. Using such database and otherfiltering options as described above, the patient and tumor specificneoepitopes may be filtered to remove those known sequences, yielding asequence set with a plurality of neoepitope sequences havingsubstantially reduced false positives.

Once the desired level of filtering for the neoepitope is accomplished(e.g., neoepitope filtered by tumor versus normal, and/or expressionlevel, and/or sub-cellular location, and/or patient specific HLA-match,and/or known variants), a further filtering step is contemplated thattakes into account the gene type that is affected by the neoepitope. Forexample, suitable gene types include cancer driver genes, genesassociated with regulation of cell division, genes associated withapoptosis, and genes associated with signal transduction. However, inespecially preferred aspects, cancer driver genes are particularlypreferred (which may span by function a variety of gene types, includingreceptor genes, signal transduction genes, transcription regulatorgenes, etc.). In further contemplated aspects, suitable gene types mayalso be known passenger genes and genes involved in metabolism.

With respect to the identification or other determination (e.g.,prediction) of a gene as being a cancer driver gene, various methods andprediction algorithms are known in the art, and are deemed suitable foruse herein. For example, suitable algorithms include MutsigCV (Nature2014, 505(7484):495-501), ActiveDriver (Mol Syst Biol 2013, 9:637),MuSiC (Genome Res 2012, 22(8):1589-1598), OncodriveClust (Bioinformatics2013, 29(18):2238-2244), OncodriveFM (Nucleic Acids Res2012,40(21):e169), OncodriveFML (Genome Biol 2016, 17(1):128), TumorSuppressor and Oncogenes (TUSON) (Cell 2013, 155(4):948-962), 20/20+(https://github.com/KarchinLab/2020plus), and oncodriveROLE(Bioinformatics (2014) 30 (17): i549-i555). Alternatively, oradditionally, identification of cancer driver genes may also employvarious sources for known cancer driver genes and their association withspecific cancers. For example, the Intogen Catalog of driver mutations(2016.5; URL: www.intogen.org) contains the results of the driveranalysis performed by the Cancer Genome Interpreter across 6,792 exomesof a pan-cancer cohort of 28 tumor types.

Nevertheless, despite filtering, it should be recognized that not allneoepitopes will be visible to the immune system as the neoepitopes alsoneed to be processed where present in a larger context (e.g., within apolytope) and presented on the MHC complex of the patient. In thatcontext, it must be appreciated that only a fraction of all neoepitopeswill have sufficient affinity for presentation. Consequently, andespecially in the context of immune therapy it should be apparent thatneoepitopes will be more likely effective where the neoepitopes areproperly processed, bound to, and presented by the MHC complexes. Viewedfrom another perspective, treatment success will be increased with anincreasing number of neoepitopes that can be presented via the MHCcomplex, wherein such neoepitopes have a minimum affinity to thepatient's HLA-type. Consequently, it should be appreciated thateffective binding and presentation is a combined function of thesequence of the neoepitope and the particular HLA-type of a patient.Therefore, HLA-type determination of the patient tissue is typicallyrequired. Most typically, the HLA-type determination includes at leastthree MHC-I sub-types (e.g., HLA-A, HLA-B, HLA-C) and at least threeMHC-II sub-types (e.g., HLA-DP, HLA-DQ, HLA-DR), preferably with eachsubtype being determined to at least 2-digit or at least 4-digit depth.However, greater depth (e.g., 6 digit, 8 digit) is also contemplated.

Once the HLA-type of the patient is ascertained (using known chemistryor in silico determination), a structural solution for the HLA-type iscalculated and/or obtained from a database, which is then used in adocking model in silico to determine binding affinity of the (typicallyfiltered) neoepitope to the HLA structural solution. As will be furtherdiscussed below, suitable systems for determination of bindingaffinities include the NetMHC platform (see e.g., Nucleic Acids Res.2008 Jul. 1; 36(Web Server issue): W509-W512.). Neoepitopes with highaffinity (e.g., less than 100 nM, less than 75 nM, less than 50 nM) fora previously determined HLA-type are then selected for therapy creation,along with the knowledge of the patient's MHC-I/II subtype.

HLA determination can be performed using various methods inwet-chemistry that are well known in the art, and all of these methodsare deemed suitable for use herein. However, in especially preferredmethods, the HLA-type can also be predicted from omics data in silicousing a reference sequence containing most or all of the known and/orcommon HLA-types. For example, in one preferred method according to theinventive subject matter, a relatively large number of patient sequencereads mapping to chromosome 6p21.3 (or any other location near/at whichHLA alleles are found) is provided by a database or sequencing machine.Most typically the sequence reads will have a length of about 100-300bases and comprise metadata, including read quality, alignmentinformation, orientation, location, etc. For example, suitable formatsinclude SAM, BAM, FASTA, GAR, etc. While not limiting to the inventivesubject matter, it is generally preferred that the patient sequencereads provide a depth of coverage of at least 5×, more typically atleast 10×, even more typically at least 20×, and most typically at least30×.

In addition to the patient sequence reads, contemplated methods furtheremploy one or more reference sequences that include a plurality ofsequences of known and distinct HLA alleles. For example, a typicalreference sequence may be a synthetic (without corresponding human orother mammalian counterpart) sequence that includes sequence segments ofat least one HLA-type with multiple HLA-alleles of that HLA-type. Forexample, suitable reference sequences include a collection of knowngenomic sequences for at least 50 different alleles of HLA-A.Alternatively, or additionally, the reference sequence may also includea collection of known RNA sequences for at least 50 different alleles ofHLA-A. Of course, and as further discussed in more detail below, thereference sequence is not limited to 50 alleles of HLA-A, but may havealternative composition with respect to HLA-type and number/compositionof alleles. Most typically, the reference sequence will be in a computerreadable format and will be provided from a database or other datastorage device. For example, suitable reference sequence formats includeFASTA, FASTQ, EMBL, GCG, or GenBank format, and may be directly obtainedor built from data of a public data repository (e.g., IMGT, theInternational ImMunoGeneTics information system, or The Allele FrequencyNet Database, EUROSTAM, URL: www.allelefrequencies.net). Alternatively,the reference sequence may also be built from individual knownHLA-alleles based on one or more predetermined criteria such as allelefrequency, ethnic allele distribution, common or rare allele types, etc.

Using the reference sequence, the patient sequence reads can now bethreaded through a de Bruijn graph to identify the alleles with the bestfit. In this context, it should be noted that each individual carriestwo alleles for each HLA-type, and that these alleles may be verysimilar, or in some cases even identical. Such high degree of similarityposes a significant problem for traditional alignment schemes. Theinventor has now discovered that the HLA alleles, and even very closelyrelated alleles can be resolved using an approach in which the de Bruijngraph is constructed by decomposing a sequence read into relativelysmall k-mers (typically having a length of between 10-20 bases), and byimplementing a weighted vote process in which each patient sequence readprovides a vote (“quantitative read support”) for each of the alleles onthe basis of k-mers of that sequence read that match the sequence of theallele. The cumulatively highest vote for an allele then indicates themost likely predicted HLA allele. In addition, it is generally preferredthat each fragment that is a match to the allele is also used tocalculate the overall coverage and depth of coverage for that allele.

Scoring may further be improved or refined as needed, especially wheremany of the top hits are similar (e.g., where a significant portion oftheir score comes from a highly shared set of k-mers). For example,score refinement may include a weighting scheme in which alleles thatare substantially similar (e.g., >99%, or other predetermined value) tothe current top hit are removed from future consideration. Counts fork-mers used by the current top hit are then re-weighted by a factor(e.g., 0.5), and the scores for each HLA allele are recalculated bysumming these weighted counts. This selection process is repeated tofind a new top hit. The accuracy of the method can be even furtherimproved using RNA sequence data that allows identification of thealleles expressed by a tumor, which may sometimes be just 1 of the 2alleles present in the DNA. In further advantageous aspects ofcontemplated systems and methods, DNA or RNA, or a combination of bothDNA and RNA can be processed to make HLA predictions that are highlyaccurate and can be derived from tumor or blood DNA or RNA. Furtheraspects, suitable methods and considerations for high-accuracy in silicoHLA typing are described in WO 2017/035392, incorporated by referenceherein.

Once patient and tumor specific neoepitopes and HLA-type are identified,further computational analysis can be performed by in silico dockingneoepitopes to the HLA and determining best binders (e.g., lowest K_(D),for example, less than 500 nM, or less than 250 nM, or less than 150 nM,or less than 50 nM), for example, using NetMHC. It should be appreciatedthat such approach will not only identify specific neoepitopes that aregenuine to the patient and tumor, but also those neoepitopes that aremost likely to be presented on a cell and as such most likely to elicitan immune response with therapeutic effect. Of course, it should also beappreciated that thusly identified HLA-matched neoepitopes can bebiochemically validated in vitro prior to inclusion of the nucleic acidencoding the epitope as payload into the virus as is further discussedbelow.

Of course, it should be appreciated that matching of the patient'sHLA-type to the patient- and cancer-specific neoepitope can be doneusing systems other than NetMHC, and suitable systems include NetMHC II,NetMHCpan, IEDB Analysis Resource (URL immuneepitope.org), RankPep,PREDEP, SVMHC, Epipredict, HLABinding, and others (see e.g., J ImmunolMethods 2011; 374:1-4). In calculating the highest affinity, it shouldbe noted that the collection of neoepitope sequences in which theposition of the altered amino acid is moved (supra) can be used.Alternatively, or additionally, modifications to the neoepitopes may beimplemented by adding N- and/or C-terminal modifications to furtherincrease binding of the expressed neoepitope to the patient's HLA-type.Thus, neoepitopes may be native as identified or further modified tobetter match a particular HLA-type. Moreover, where desired, binding ofcorresponding wild type sequences (i.e., neoepitope sequence withoutamino acid change) can be calculated to ensure high differentialaffinities. For example, especially preferred high differentialaffinities in MHC binding between the neoepitope and its correspondingwild type sequence are at least 2-fold, at least 5-fold, at least10-fold, at least 100-fold, at least 500-fold, at least 1000-fold,etc.).

Binding affinity and particularly differential binding affinity may alsobe determined in vitro using various systems and methods. For example,antigen presenting cells of a patient or cells with matched HLA-type canbe transfected with a nucleic acid (e.g., viral, plasmid, linear DNA,RNA, etc.) to express one or more neoepitopes using constructs asdescribed in more detail below. Upon expression and antigen processing,the neoepitopes can then be identified in the MHC complex on the outsideof the cell, either using specific binders to the neoepitope or using acell based system (e.g., PBMC of the patient) in which T cell activationor cytotoxic NK cell activity can be observed in vitro. Neoepitopes withdifferential activity (elicit a stronger signal or immune response ascompared to the corresponding wild type epitope) will then be selectedfor therapy creation.

Recombinant Nucleic Acids/Polytopes

Upon proper selection of filtered neoepitopes (or tumor associatedantigens or tumor specific antigens), a recombinant nucleic acid can beconstructed that forms the basis of all downstream vaccine compositions.Most typically, the desired nucleic acid sequences (for expression fromvirus infected cells) are under the control of appropriate regulatoryelements well known in the art. As will also be readily appreciated, thechoice of regulatory elements will be dictated by the system in whichthe recombinant nucleic acid is to be expressed. Therefore, suitableregulatory elements include constitutively active or inducible bacterialand yeast promoters (and associated inducer and/or repressor sequenceswhere desired), as well as eukaryotic (and preferably mammal/human)promoter sequences. For example, where the recombinant nucleic acid isused in a DNA vaccine, suitable promoter elements include constitutivestrong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoter). Onthe other hand, where the recombinant nucleic acid is part of a viralexpression vector, contemplated promoters also include induciblepromoters, particularly where induction conditions are typical for atumor microenvironment. For example, inducible promoters include thosesensitive to hypoxia and promoters that are sensitive to TGF-β or IL-8(e.g., via TRAF, JNK, Erk, or other responsive elements promoter). Inother examples, suitable inducible promoters include thetetracycline-inducible promoter, the myxovirus resistance 1 (Mx1)promoter, etc.

Similarly, where the recombinant nucleic acid is used to generate abacterial and/or yeast vaccine in which the bacterium or yeast expressesthe neoepitope or other therapeutic antigen, suitable promoters includestrong constitutive or inducible bacterial and yeast promoters. Forexample, suitable bacterial promoters for expression of antigens/apolytope include the T7 promoter, the Tac promoter, the BAD promoter,the Trc promoter, etc. Likewise, yeast contemplated yeast promotersinclude the AOX1 promoter, the GAL promoter, the GDS promoter, the ADHpromoter, etc.

In this context, it should be appreciated that the inventors havediscovered that the manner of neoepitope arrangement andrational-designed trafficking of the neoepitopes can have a substantialimpact on the efficacy of various immune therapeutic compositions as isfurther described in more detail below. For example, single neoepitopescan be expressed individually from the respective recombinant constructsthat are delivered as a single plasmid, viral expression construct, etc.Alternatively, multiple neoepitopes can be separately expressed fromindividual promoters to form individual mRNA that are then individuallytranslated into the respective neoepitopes, or from a single mRNAcomprising individual translation starting points for each neoepitopesequence (e.g., using 2A or IRES signals). Notably, while sucharrangements are generally thought to allow for controlled delivery ofproper neoepitope peptide, efficacy of such expression systems has beenless than desirable (data not shown).

In contrast, where multiple neoepitopes were expressed from a singletranscript to so form a single transcript that is then translated into asingle polytope (i.e., polypeptide with a series of concatemericallylinked neoepitopes, optionally with intervening linker sequences)expression, processing, and antigen presentation was found to beeffective. Notably, the expression of polytopes requires processing bythe appropriate proteases (e.g., proteasome, endosomal proteases,lysosomal proteases) within a cell to yield the neoepitope sequences,and polytopes led to improved antigen processing and presentation formost neoepitopes as compared to expression of individual neoepitopes,particularly where the individual neoepitopes had a relatively shortlength (e.g., less than 25 amino acids; results not shown). Moreover,such approach also allows rational design of protease sensitive sequencemotifs between the neoepitope peptide sequences to so assure or avoidprocessing by specific proteases as the proteasome, endosomal proteases,and lysosomal proteases have distinct cleavage preferences. Therefore,polytopes may be designed that include not only linker sequences tospatially separate neoepitopes, but also sequence portions (e.g.,between 3-15 amino acids) that will be preferentially cleaved by aspecific protease.

Therefore, the inventors contemplate recombinant nucleic acids andexpression vectors (e.g., viral expression vectors) that comprise anucleic acid segment that encodes a polytope wherein the polytope isoperably coupled to a desired promoter element, and wherein individualneoepitopes are optionally separated by a linker and/or proteasecleavage or recognition sequence. For example, FIG. 1 exemplarilyillustrates various contemplated arrangements for neoepitopes forexpression from an adenoviral expression system (here: AdV5, withdeletion of E1 and E2b genes). Here, Construct 1 exemplarily illustratesa neoepitope arrangement that comprises eight neoepitopes (‘minigene’)with a total length of 15 amino acids in concatemeric series withoutintervening linker sequences, while Construct 2 shows the arrangement ofConstruct 1 but with inclusion of nine amino acid linkers between eachneoepitope sequence. Of course, and as already noted above, it should berecognized that the exact length of the neoepitope sequence is notlimited to 15 amino acids, and that the exact length may varyconsiderably. However, in most cases, where neoepitope sequences ofbetween 8-12 amino acids are flanked by additional amino acids, thetotal length will typically not exceed 25 amino acids, or 30 aminoacids, or 50 amino acids. Likewise, it should be noted that while FIG. 1denotes G-S linkers, various other linker sequences are also suitablefor use herein. Such relatively short neoepitopes are especiallybeneficial where presentation of the neoepitope is intended to be viathe MHC-I complex.

In this context, it should be appreciated that suitable linker sequenceswill provide steric flexibility and separation of two adjacentneoepitopes. However, care must be taken to as to not choose amino acidsfor the linker that could be immunogenic/form an epitope that is alreadypresent in a patient. Consequently, it is generally preferred that thepolytope construct is filtered once more for the presence of epitopesthat could be found in a patient (e.g., as part of normal sequence ordue to SNP or other sequence variation). Such filtering will apply thesame technology and criteria as already discussed above.

Similarly, Construct 3 exemplarily illustrates a neoepitope arrangementthat includes eight neoepitopes in concatemeric series withoutintervening linker sequences, and Construct 4 shows the arrangement ofConstruct 3 with inclusion of nine amino acid linkers between eachneoepitope sequence. As noted above, it should be recognized that theexact length of such neoepitope sequences is not limited to 25 aminoacids, and that the exact length may vary considerably. However, in mostcases, where neoepitope sequences of between 14-20 amino acids areflanked by additional amino acids, the total length will typically notexceed 30 amino acids, or 45 amino acids, or 60 amino acids. Likewise,it should be noted that while FIG. 1 denotes G-S linkers for theseconstructs, various other linker sequences are also suitable for useherein. Such relatively long neoepitopes are especially beneficial wherepresentation of the neoepitope is intended to be via the MHC-II complex.

In this example, it should be appreciated that the 15-aa minigenes areMHC Class I targeted tumor mutations selected with 7 amino acids ofnative sequence on either side, and that the 25-aa minigenes are MHCClass II targeted tumor mutations selected with 12 amino acids of nativesequence on either side. The exemplary 9 amino acid linkers are deemedto have sufficient length such that “unnatural” MHC Class I epitopeswill not form between adjacent minigenes. Polytope sequences tended tobe processed and presented more efficiently than single neoepitopes(data not shown), and addition of amino acids beyond 12 amino acids forMHC-I presentation and addition of amino acids beyond 20 amino acids forMHC-I presentation appeared to allow for somewhat improved proteaseprocessing.

To maximize the likelihood that customized protein sequences remainintracellular for processing and presentation by the HLA complex,neoepitope sequences may be arranged in a manner to minimize hydrophobicsequences that may direct trafficking to the cell membrane or into theextracellular space. Most preferably, hydrophobic sequence or signalpeptide detection is done either by comparison of sequences to a weightmatrix (see e.g., Nucleic Acids Res. 1986 Jun. 11; 14(11): 4683-4690) orby using neural networks trained on peptides that contain signalsequences (see e.g., Journal of Molecular Biology 2004, Volume 338,Issue 5, 1027-1036). FIG. 2 depicts an exemplary scheme of arrangementselection in which a plurality of polytope sequences are analyzed. Here,all positional permutations of all neoepitopes are calculated to producea collection of arrangements. This collection is then processed througha weight matrix and/or neural network prediction to generate a scorerepresenting the likelihood of presence and/or strength of hydrophobicsequences or signal peptides. All positional permutations are thenranked by score, and the permutation(s) with a score below apredetermined threshold or lowest score for likelihood of presenceand/or strength of hydrophobic sequences or signal peptides is/are usedto construct a customized neoepitope expression cassette.

With respect to the total number of neoepitope sequences in a polytopeit is generally preferred that the polytope comprise at least two, or atleast three, or at least five, or at least eight, or at least tenneoepitope sequences. Indeed, the payload capacity of the host organismof the recombinant DNA is generally contemplated the limiting factor,along with the availability of filtered and appropriate neoepitopes. Forexample, adenoviral expression vectors, and particularly Adv5 areespecially preferred as such vectors can accommodate up to 14 kb inrecombinant payload. Likewise, bacterial and yeast systems canaccommodate even larger payloads, typically in excess of 50 kb. On theother hand, where the recombinant DNA is used in a DNA vaccine, suitablesizes will typically range between 5 kb and 20 kb.

In still further contemplated aspects of the inventive subject matter,it should be noted that the neoepitopes/polytopes can be directedtowards a specific sub-cellular compartment (e.g., cytosol, endosome,lysosome), and with that, towards a particular MHC presentation type.Such directed expression, processing, and presentation is particularlyadvantageous as contemplated compositions may be prepared that direct animmune response towards a CD8⁺ type response (where the polytope isdirected to the cytoplasmic space) or towards a CD4⁺ type response(where the polytope is directed to the endosomal/lysosomal compartment).Moreover, it should be recognized that polytopes that would ordinarilybe presented via the MHC-I pathway can be presented via the MHC-IIpathway (and thereby mimic cross-presentation of neoepitopes).Therefore, it should be appreciated that neoepitope and polytopesequences may be designed and directed to one or both MHC presentationpathways using suitable sequence elements. With respect to routing theso expressed neoepitopes to the desired MHC-system, it is noted that theMHC-I presented peptides will typically arise from the cytoplasm viaproteasome processing and delivery through the endoplasmic reticulum.Thus, expression of the epitopes intended for MHC-I presentation willgenerally be directed to the cytoplasm as is further discussed in moredetail below. On the other hand, MHC-II presented peptides willtypically arise from the endosomal and lysosomal compartment viadegradation and processing by acidic proteases (e.g., legumain,cathepsin L and cathepsin S) prior to delivery to the cell membrane.

Moreover, it is contemplated that proteolytic degradation of thepolytope can also be enhanced using various methods, and especiallycontemplated methods include addition of a cleavable or non-cleavableubiquitin moiety to the N-terminus, and/or placement of one or moredestabilizing amino acids (e.g., N, K, C, F, E, R, Q) to the N-terminusof the polytope where the presentation is directed towards MHC-I. On theother hand, where presentation is directed towards MHC-II, cleavagesites for particular endosomal or lysosomal proteases can be engineeredinto the polytope to so facilitate of promote antigen processing.

Therefore, in contemplated aspects of the inventive subject matter,signal and/or leader peptides may be used for trafficking neoepitopesand/or polytopes to the endosomal and lysosomal compartment, or forretention in the cytoplasmic space. For example, where the polytope isto be exported to the endosomal and lysosomal compartment, a leaderpeptide such as the CD1b leader peptide may be employed to sequester the(nascent) protein from the cytoplasm. Additionally, or alternatively,targeting presequences and/or targeting peptides can be employed. Thepresequences of the targeting peptide may be added to the N-terminusand/or C-terminus and typically comprise between 6-136 basic andhydrophobic amino acids. In case of peroxisomal targeting, the targetingsequence may be at the C-terminus. Other signals (e.g., signal patches)may be used and include sequence elements that are separate in thepeptide sequence and become functional upon proper peptide folding. Inaddition, protein modifications like glycosylations can inducetargeting. Among other suitable targeting signals, the inventorscontemplate peroxisome targeting signal 1 (PTS1), a C-terminaltripeptide, and peroxisome targeting signal 2 (PTS2), which is anonapeptide located near the N-terminus.

In addition, sorting of proteins to endosomes and lysosomes may also bemediated by signals within the cytosolic domains of the proteins,typically comprising short, linear sequences. Some signals are referredto as tyrosine-based sorting signals and conform to the NPXY or YXXØconsensus motifs. Other signals known as dileucine-based signals fit[DE]XXXL[LI] or DXXLL consensus motifs. All of these signals arerecognized by components of protein coats peripherally associated withthe cytosolic face of membranes. YXXØ and [DE]XXXL[LI] signals arerecognized with characteristic fine specificity by the adaptor protein(AP) complexes AP-1, AP-2, AP-3, and AP-4, whereas DXXLL signals arerecognized by another family of adaptors known as GGAs. Also FYVE domaincan be added, which has been associated with vacuolar protein sortingand endosome function. In still further aspects, endosomal compartmentscan also be targeted using human CD1 tail sequences (see e.g.,Immunology, 122, 522-531). For example, lysosomal targeting can beachieved using a LAMP1-TM (transmembrane) sequence, while recyclingendosomes can be targeted via the CD1a tail targeting sequence, andsorting endosomes can be targeted via the CD1c tail targeting sequenceas is shown in more detail further below.

Trafficking to or retention in the cytosolic compartment may notnecessarily require one or more specific sequence elements. However, inat least some aspects, N- or C-terminal cytoplasmic retention signalsmay be added, including a membrane-anchored protein or a membrane anchordomain of a membrane-anchored protein such that the protein is retainedin the cell facing the cytosol. For example, membrane-anchored proteinsinclude SNAP-25, syntaxin, synaptoprevin, synaptotagmin, vesicleassociated membrane proteins (VAMPs), synaptic vesicle glycoproteins(SV2), high affinity choline transporters, Neurexins, voltage-gatedcalcium channels, acetylcholinesterase, and NOTCH.

In still further contemplated aspects of the inventive subject matter,the polytope may also comprise one or more transmembrane segments thatwill direct the neoepitope after processing to the outside of the cellmembrane to so be visible to immune competent cells. There are numeroustransmembrane domains known in the art, and all of those are deemedsuitable for use herein, including those having a single alpha helix,multiple alpha helices, alpha/beta barrels, etc. For example,contemplated transmembrane domains can comprise comprises thetransmembrane region(s) of the alpha, beta, or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160,CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4,CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a,LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1,ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CEACAM1, CRTAM,Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108),SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, orPAG/Cbp. Where a fusion protein is desired, it is contemplated that therecombinant chimeric gene has a first portion that encodes thetransmembrane region(s), wherein the first portion is cloned in framewith a second portion that encodes the inhibitory protein. It should benoted that such presentation will not result in MHC-complex presentationand as such provides a neoepitope presentation independent of MHC/T-cellreceptor interaction, which may further open additional avenues forimmune recognition and trigger antibody production against theneoepitopes.

Alternatively, or additionally, the polytope may also be designed toinclude signal sequences for protein export of one or more neoepitope tothereby force a transfected cell to produce and secrete one or moreneoepitopes. For example, the SPARC leader sequence may be added to aneoepitope or polytope sequence, leading to in vivo secretion of theneoepitope or polytope sequence into the extracellular space.Advantageously, such secreted neoepitopes or polytopes are then taken upby immune competent cells, and especially antigen presenting cells anddendritic cells that in turn process and display the neoepitopes,typically via MHC-II pathways.

In still further contemplated aspects, the polytope may also be designedas a chimeric polytope that includes at least a portion of, and moretypically an entire tumor associated antigen (e.g., CEA, PSMA, PSA,MUC1, AFP, MAGE, HER2, HCC1, p62, p90, etc.). Most notably, tumorassociated antigens are generally processed and presented via the MHC-IIpathway. Therefore, instead of using compartment specific signalsequences and/or leader sequences, the processing mechanism for tumorassociated antigens can be employed for MHC-II targeting.

Therefore, it should be appreciated that immune therapeutic compositionsmay be prepared that can deliver one or more neoepitopes to varioussub-cellular locations, and with that generate distinct immuneresponses. For example, Prior Art FIG. 3 schematically illustrates ascenario where the polytope is predominantly processed in the proteasomeof the cytoplasm and presented via the MHC-I complex, which isrecognized by the T-cell receptor of a CD8⁺ T-cell. Consequently,targeting polytope processing to the cytosolic compartment will skew theimmune response towards a CD8⁺ type response. On the other hand, PriorArt FIG. 4 schematically illustrates a scenario where the polytope ispredominantly processed in the endosomal compartment and presented viathe MHC-II complex, which is recognized by the T-cell receptor of a CD4⁺T-cell. Consequently, targeting polytope processing to the endosomal orlysosomal compartment will skew the immune response towards a CD4⁺ typeresponse. In addition, it should be appreciated that such targetingmethods allow for specific delivery of a polytope or neoepitope peptideto an MHC subtype having the highest affinity with the peptide, even ifthat peptide would otherwise not be presented by that MHC subtype.Therefore, and as noted earlier, peptides for MHC-I presentation willgenerally be designed to have 8-12 amino acids (plus additional aminoacids for flexibility in protease processing), while peptides for MHC-IIpresentation will be designed to have 14-20 amino acids (plus additionalamino acids for flexibility in protease processing). In the examplesbelow, further amino acids were added to allow for processingflexibility in the cytoplasmic, proteasome, or endosomal compartments.

In still further contemplated aspects of the inventive subject matter,it should be noted that trafficking modes of the neoepitope or polytopemay be combined to accommodate one or more specific purposes. Forexample, sequential administration of the same neoepitopes or polytopewith different targeting may be particularly beneficial in a prime-boostregimen where in a first administration the patient in inoculated with arecombinant virus to infect the patients cells, leading to antigenexpression, processing, and presentation (e.g., predominantly MHC-Ipresentation) that will result in a first immune response originatingfrom within a cell. The second administration of the same neoepitopesbound to albumin may then be employed as a boost as the so deliveredprotein is taken up by antigen presenting cells, leading in most casesto a distinct antigen presentation (e.g., predominantly MHC-IIpresentation). Where the same neoepitopes or polytope is trafficked tothe cell surface for cell surface bound MHC-independent presentation,ADCC responses or NK mediated cell killing may be promoted. In stillfurther contemplated aspects, and as illustrated in the examples below,immunogenicity of neoepitopes may be enhanced by cross presentation orMHC-II directed presentation. Notably, as cancer cell neoepitopes aretypically internally generated and recycled, and with thatpreferentially presented via the MHC-I system, contemplated systems andmethods now allow for presentation of such neoepitopes via MHC-II, whichmay be more immunogenic as is shown in more detail below. In addition,multiple and distinct trafficking of the same neoepitopes or polytopesmay advantageously increase or supplement an immune response due to thestimulation of various and distinct components of the cellular andhumoral immune system.

Of course, it should be appreciated that multiple and distincttrafficking of the same neoepitopes or polytopes may be achieved innumerous manners. For example, differently trafficked neoepitopes orpolytopes may be administered separately using the same (e.g., viralexpression vector) or different (e.g., viral expression vector andalbumin bound) modality. Similarly, and especially where the therapeuticagent is an expression system (e.g., viral or bacterial), therecombinant nucleic acid may include two distinct portions that encodethe same, albeit differently trafficked neoepitope or polytope (e.g.,first portion trafficked to first location (e.g., cytosol or endosomalor lysosomal), second portion trafficked to a second, distinct location(e.g., cytosol or endosomal or lysosomal, secreted, membrane bound)).Likewise, a first administration may employ viral delivery of cytoplasmtargeted neoepitopes or polytope, while a second administration istypically at least a day, two days, four days, a week, or two weeksafter the first administration and may employ viral delivery ofendosomal or lysosomal targeted or secreted neoepitopes or polytope.

In addition, it should be appreciated that where the recombinant nucleicacid is used in a DNA, bacterial, or yeast vaccine, the manner of uptakeof these modalities will at least partially dictate intracellulartrafficking. Most typically, DNA, bacterial, or yeast vaccines are takenup by endocytotic or related processes and as such will bepreferentially routed to the endosomal or lysosomal compartments. Suchrouting can be further enhanced (at least in the case of DNA vaccines)using appropriate trafficking signals as already described above orcounteracted by use of a cytoplasmic retention sequence. However, otherembodiments, it should be appreciated that polytopes delivered via DNA,bacterial, or yeast vaccines need not have a trafficking signal at all.Such polytopes will then preferentially processed/presented via theMHC-II system.

Additionally, it is contemplated that the expression construct, andespecially the recombinant viral expression vector or DNA plasmid for aDNA vaccine, may further encode at least one, more typically at leasttwo, even more typically at least three, and most typically at leastfour co-stimulatory molecules to enhance the interaction between theinfected cells (e.g., antigen presenting cells) and T-cells. Forexample, suitable co-stimulatory molecules include CD80, CD86, CD30,CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, whileother stimulatory molecules with less defined (or understood) mechanismof action include GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, LFA3,and members of the SLAM family. However, especially preferred moleculesfor coordinated expression with the cancer-associated sequences includeCD80 (B7-1), CD86 (B7-2), CD54 (ICAM-1) and CD11 (LFA-1). In addition toco-stimulatory molecules, the inventors also contemplate that one ormore cytokines or cytokine analogs may be expressed from the recombinantnucleic acid, and especially preferred cytokines and cytokine analogsinclude IL-2, IL-15, and IL-a5 superagonist (ALT-803). Moreover, itshould be appreciated that expression of the co-stimulatory moleculesand/or cytokines will preferably be coordinated such that theneoepitopes or polytope are expressed contemporaneously with one or moreco-stimulatory molecules and/or cytokines. Thus, it is typicallycontemplated that the co-stimulatory molecules and/or cytokines areproduced from a single transcript (which may or may not include thesequence portion encoding the polytope), for example, using an internalribosome entry site or 2A sequence, or from multiple transcripts.

Likewise, it is contemplated that the viral vector may also include asequence portion that encodes one or more peptide ligands that bind to acheckpoint receptor. Most typically, binding will inhibit or at leastreduce signaling via the receptor, and particularly contemplatedreceptors include CTLA-4 (especially for CD8⁺ cells), PD-1 (especiallyfor CD4⁺ cells), TIM1 receptor, 2B4, and CD160. For example, suitablepeptide binders can include antibody fragments and especially scFv, butalso small molecule peptide ligands (e.g., isolated via RNA display orphage panning) that specifically bind to the receptors. Once more, itshould be appreciated that expression of the peptide molecules willpreferably be coordinated such that the neoepitopes or polytope areexpressed contemporaneously with one or more of the peptide ligands.Thus, it is typically contemplated that the peptide ligands are producedfrom a single transcript (which may or may not include the sequenceportion encoding the polytope), for example, using an internal ribosomeentry site or 2A sequence, or from multiple transcripts.

It should be appreciated that all of the above noted co-stimulatorygenes and genes coding for inhibitory proteins that interferewith/down-regulate checkpoint inhibition are well known in the art, andsequence information of these genes, isoforms, and variants can beretrieved from various public resources, including sequence data basesaccessible at the NCBI, EMBL, GenBank, RefSeq, etc. Moreover, while theabove exemplary stimulating molecules are preferably expressed in fulllength form as expressed in human, modified and non-human forms are alsodeemed suitable so long as such forms assist in stimulating oractivating T-cells. Therefore, muteins, truncated forms and chimericforms are expressly contemplated herein.

Consequently, contemplated expression constructs will preferably includea sequence portion that encodes one or more polytopes, wherein at leastone, and more typically at least two, or all of the polytopes willinclude a trafficking signal that will result in preferentialtrafficking of the polytope to at least one, and more typically at leasttwo different sub-cellular locations.

For example, the first polytope may be directed towards the cytoplasm(and may include an additional cleavable or non-cleavable ubiquitin)while the second polytope may be directed towards the endosomal orlysosomal compartment. Or the first polytope may be directed towards theendosomal or lysosomal compartment while the second polytope may bedirected towards the cell membrane or be secreted. As noted before, theencoded polytope will comprise at least two neoepitopes, optionallyseparated by a linker. Moreover, such contemplated expression constructswill also include a sequence portion that encodes one or moreco-stimulatory molecules and/or cytokines, and may also include one ormore inhibitory proteins that interfere with/down-regulate checkpointinhibition. Most typically, the expression construct will also includeregulatory sequences operably coupled to the above sequence portions todrive contemporaneous expression of the polytope and the co-stimulatorymolecules, cytokines, and/or inhibitory proteins. Suitable promoterelements are known in the art, and especially preferred promotersinclude the constitutive and inducible promoters discussed above.

Vaccine Compositions

Upon identification of desired neoepitopes, one or more immunetherapeutic agents may be prepared using the sequence information of theneoepitopes, preferably configured as a polytope as described above.Preferably, the immune therapeutic agents include at least two of a DNAvaccine that includes a recombinant nucleic acid that encodes at leastone antigen (and more typically at least two, three, four, or moreantigens) that is present in the tumor, a bacterial vaccine in which abacterium expresses at least one antigen (and more typically at leasttwo, three, four, or more antigens) that is present in the tumor, ayeast vaccine in which a bacterium expresses at least one antigen (andmore typically at least two, three, four, or more antigens) that ispresent in the tumor, and a viral vaccine that comprises a viralexpression vector that includes a recombinant nucleic acid that encodesat least one antigen (and more typically at least two, three, four, ormore antigens) that is present in the tumor.

With respect to recombinant nucleic acids for expression and DNAvaccination systems it is contemplated that the recombinant nucleic acidmay be an RNA or a DNA. With respect to the use of RNA, DNA, or otherrecombinant vectors that lead to the expression of the tumor antigensand/or neoepitopes, especially contemplated nucleic acids includeplasmid vectors that may be supercoiled, coiled, relaxed, or evenlinearized. For example, and among other suitable choices, contemplatedvectors include vectors used in cloning one or more sequence portionsused in the preparation of the viral expression vector. Thus, especiallycontemplated vectors include transfer or shuttle vectors, and variousgeneral cloning vectors (e.g., having a bacterial origin of replication,a selection marker (e.g., antibiotic resistance or fluorescent protein),and a multiple cloning site). Suitable vectors are well known in the artand are typically based on a plasmid with replicative capability inbacteria for cloning and production of substantial quantities. Propervector selection may further be determined by its particular use (e.g.,shuttle vector for adenovirus, lentivirus, or baculovirus, etc.), choiceof inducible or constitutive promoter (e.g., CMV, UbC), choice ofpermanent or transient expression, manner of transfection (e.g.,lipofection, electroporation, etc.), capacity for the recombinantpayload, etc.

It should still further be noted that plasmids may be methylated orunmethylated, which may be controlled by directed enzymatic in vitroreactions or more simply by in vivo replication of the plasmid in amethylation competent or a methylation deficient host. Still further, itshould be appreciated that the plasmid may further have one or morenucleic acid portions that are known to trigger an innate immuneresponse (e.g., CpG islands or other sequence motifs that interact withToll-like receptors such as TLR3, TLR 7, TLR 8, TLR 9, RIG-I-likereceptors, STING, and/or intracellular DNA sensors such as NLRP3/CIAS1).

Most typically, and as already noted above, contemplated plasmids andother nucleic acids will include one or more sequence elements thatencode the preferably patient- and tumor specific neoepitopes or thepolytope, most preferably operably coupled to regulatory elements thatpermit or drive the expression of the neoepitope or polytope ineukaryotic cells, and especially mammalian cells (e.g., human).Moreover, it should be noted that the neoepitope or polytope may includethe trafficking signals for routing the peptide to the desiredsub-cellular location as also discussed herein. Therefore, especiallypreferred plasmids include plasmids used in the production of a viralexpression vector, and as such will already include all regulatoryelements needed for expression and/or trafficking of the polytope in amammalian cell. Thus cloning vectors and shuttle vectors are especiallypreferred.

As will be appreciated, plasmids contemplated herein may be administeredin numerous manners known in the art, and suitable delivery modesinclude injection (intramuscular, intravenous, intradermal), deliveryvia gene gun or other ballistic transfer, or by liposome mediatedtransfer. Therefore, contemplated compositions will particularly includeinjectable formulations comprising nucleic acid lipoplexes and otherDNA-lipid or DNA lipoprotein complexes. Advantageously, the choice ofdelivery may be used to polarize the immune response towards either Th1(via injection using saline) or Th2 (via gene gun delivery) response asdescribed elsewhere (see e.g., J Immunol Mar. 1, 1997, 158 (5)2278-2284). In especially preferred aspects, vaccination with DNA willpreferably be performed by intravenous injection as is discussed in moredetail below, however, other routes (including intramuscular,intradermal, subcutaneous, intra-arterial) are also deemed suitable foruse herein, while administration of the viral expression vector(typically using a recombinant virus) will preferably be performed bysubcutaneous injection.

Without wishing to be bound by any specific theory or hypothesis, it isbelieved that the use of plasmids and other ‘naked’ nucleic acids (e.g.,linear DNA RNA) will provide a first opportunity to generate an immuneresponse that is both non-specific and specific to the patient'sneoepitope. Non-specific reactions are deemed to arise from a componentof the innate immune response against foreign nucleic acids, forexample, where the nucleic acid is unmethylated (e.g., via TLR-9). Inaddition, expression of the neoepitope or polytope in the cellstransfected with the plasmid will also promote adaptive immuneresponses.

In addition to the use of DNA vaccination, contemplated plasmids mayalso be used in the production of a viral or yeast expression vectorthat can be employed to produce a recombinant virus (e.g., lentivirus,adenovirus) or yeast for subsequent administration to the patient. Wherethe plasmid is used for production of a yeast or viral expressionsystem, all known expression systems are deemed suitable for use herein.For example, suitable materials and protocols can be found in thenon-profit plasmid repository Addgene or in the AdEasy adenoviral vectorsystem (commercially available from Agilent). Similarly, there arenumerous yeast expression systems known in the art and all of those aredeemed suitable for use herein. Such second administration can be viewedas a boost regimen to the DNA vaccination as the mammal was alreadyprimed by the plasmid vector. Of course, it should be recognized thatwhere the prime vaccination was a DNA vaccination with a plasmid asdescribed above, the boost may use various alternate formats, includinga vaccination with the neoepitope peptides or polytope using moreconventional vaccine formulations. Further suitable DNA vaccines aredescribed, for example, in US 2014/0178438.

With respect to bacterial expression and vaccination systems it iscontemplated that all bacterial strains are deemed suitable, andespecially include species from Salmonella, Clostridium, Bacillus,Lactobacillus, Bifidobacterium, etc., particularly where such strainsare non-pathogenic, genetically engineered to have reduced toxicity,and/or were irradiated prior to administration. Historically, mostbacteria strains have been deemed unsuitable for introducing into theblood stream or transplanting into an organ or tissue, as most bacteriaexpress lipopolysaccharides that trigger immune responses and causeendotoxic responses, which can lead potentially fatal sepsis (e.g.,CD-14 mediated sepsis) in patients. Thus, one especially preferredbacterial strain is based on a genetically modified bacterium whichexpresses endotoxins at a level low enough not to cause an endotoxicresponse in human cells and/or insufficient to induce a CD-14 mediatedsepsis when introduced to the human body.

One preferred bacterial species is a genetically modified Escherichiacoli (E. coli) strain due to its fast growth (e.g., one complete cellcycle in 20 min) and availability of many strains optimized for proteinoverexpression upon induction (e.g., lac promoter induction with IPTG,etc.). Most typically, the genetic modification will reduce or removeproduction of most lipopolysaccharide components leading to endotoxicresponse. For example, one exemplary bacteria strain with modifiedlipopolysaccharide synthesis includes ClearColi® BL21(DE3)electrocompetent cells. This bacterial strain is BL21 with a genotypeF-ompT hsdSB (rB− mB−) gal dcm lon λ(DE3 [lacI lacUV5-T7 gene 1 ind1sam7 nin5]) msbA148 ΔgutQΔkdsD ΔlpxLΔlpxMΔpagPΔlpxPAeptA. In thiscontext, it should be appreciated that several specific deletionmutations (ΔgutQ ΔkdsD ΔlpxL ΔlpxMΔpagPΔlpxPAeptA) encode themodification of LPS to Lipid IV_(A), while one additional compensatingmutation (msbA148) enables the cells to maintain viability in thepresence of the LPS precursor lipid IVA. These mutations result in thedeletion of the oligosaccharide chain from the LPS. More specifically,two of the six acyl chains are deleted. The six acyl chains of the LPSare the trigger which is recognized by the Toll-like receptor 4 (TLR4)in complex with myeloid differentiation factor 2 (MD-2), causingactivation of NF-κB and production of proinflammatory cytokines. LipidIV_(A), which contains only four acyl chains, is not recognized by TLR4and thus does not trigger the endotoxic response. While electrocompetentBL21 bacteria is provided as an example, the inventors contemplates thatthe genetically modified bacteria can be also chemically competentbacteria.

Alternatively, the inventors also contemplate that the patient's ownendosymbiotic bacteria can be used as a vehicle to express humandisease-related antigens in vivo to elicit immune response at leastlocally. As used herein, the patient's endosymbiotic bacteria refersbacteria residing in the patient's body regardless of the patient'shealth condition without invoking any substantial immune response. Thus,it is contemplated that the patient's endosymbiotic bacteria is a normalflora of the patient. For example, the patient's endosymbiotic bacteriamay include E. coli or Streptococcus that can be commonly found in humanintestine or stomach. In these embodiments, patient's own endosymbioticbacteria can be obtained from the patient's biopsy samples from aportion of intestine, stomach, oral mucosa, or conjunctiva, or in fecalsamples. The patient's endosymbiotic bacteria can then be cultured invitro and transfected with nucleotides encoding human disease-relatedantigen(s).

Therefore, it should be appreciated that the bacteria used in themethods presented herein may be from a strain that produces LPS, or thatare genetically engineered to have reduced or abrogated expression ofone or more enzymes leading to the formation of LPS that is recognizedby a TLR, and particularly TLR4. Most typically, such bacteria will begenetically modified to express in an inducible manner at least onehuman disease-related antigen for immunotherapy. Among other options,induction of expression may be done with synthetic compounds that arenot ordinarily found in a mammal (e.g., IPTG, substituted benzenes,cyclohexanone-related compounds) or with compounds that naturally occurin a mammal (e.g., sugars (including 1-arabinose, 1-rhamnose, xylose,and sucrose), ε-caprolactam, propionate, or peptides), or induction maybe under the control of one or more environmental factors (e.g.,temperature or oxygen sensitive promoter).

Contemplated recombinant nucleic acids that encode the tumor antigens orthe polytope can be inserted into an expression vector that has aspecific promoter (e.g., inducible promoter, etc.) to drive expressionof the antigens or polytope in the bacterium. The vector is thentransfected into the bacterium (e.g., ClearColi® BL21(DE3)electrocompetent cells) following conventional methods, or any othertype of competent bacterium expressing low endotoxin level that isinsufficient to induce a CD-14 mediated sepsis when introduced to thehuman body), or to patient's own endosymbiotic bacterium that isoptionally cultured in vitro before transformation as described above.

With respect to yeast expression and vaccination systems it iscontemplated that all known yeast strains are deemed suitable for useherein. However, it is preferred that the yeast is a recombinantSaccharomyces train that is genetically modified with a nucleic acidconstruct as discussed above that leads to expression of at least one ofthe tumor antigens to thereby initiate an immune response against thetumor. Nevertheless, it is noted that any yeast strain can be used toproduce a yeast vehicle of the present invention. Yeasts are unicellularmicroorganisms that belong to one of three classes: Ascomycetes,Basidiomycetes and Fungi Imperfecti. One consideration for the selectionof a type of yeast for use as an immune modulator is the pathogenicityof the yeast. In preferred embodiments, the yeast is a non-pathogenicstrain such as Saccharomyces cerevisiae as non-pathogenic yeast strainsminimize any adverse effects to the individual to whom the yeast vehicleis administered. However, pathogenic yeast may also be used if thepathogenicity of the yeast can be negated using pharmaceuticalintervention.

For example, suitable genera of yeast strains include Saccharomyces,Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula,Schizosaccharomyces and Yarrowia. In one aspect, yeast genera areselected from Saccharomyces, Candida, Hansenula, Pichia orSchizosaccharomyces, and in a preferred aspect, Saccharomyces is used.Species of yeast strains that may be used in the invention includeSaccharomyces cerevisiae, Saccharomyces carlsbergensis, Candidaalbicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii,Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha,Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianusvar. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomycespombe, and Yarrowia lipolytica.

It should further be appreciated that a number of these species includea variety of subspecies, types, subtypes, etc. that are intended to beincluded within the aforementioned species. In one aspect, yeast speciesused in the invention include S. cerevisiae, C. albicans, H. polymorpha,P. pastoris and S. pombe. S. cerevisiae is useful due to it beingrelatively easy to manipulate and being “Generally Recognized As Safe”or “GRAS” for use as food additives (GRAS, FDA proposed Rule 62FR18938,Apr. 17, 1997). Therefore, the inventors particularly contemplate ayeast strain that is capable of replicating plasmids to a particularlyhigh copy number, such as a S. cerevisiae cir strain. The S. cerevisiaestrain is one such strain that is capable of supporting expressionvectors that allow one or more target antigen(s) and/or antigen fusionprotein(s) and/or other proteins to be expressed at high levels. Inaddition, any mutant yeast strains can be used in the present invention,including those that exhibit reduced post-translational modifications ofexpressed target antigens or other proteins, such as mutations in theenzymes that extend N-linked glycosylation.

Expression of contemplated antigens/neoepitopes in yeast can beaccomplished using techniques known to those skilled in the art. Mosttypically, a nucleic acid molecule encoding at least neoepitope or otherprotein is inserted into an expression vector such manner that thenucleic acid molecule is operatively linked to a transcription controlsequence to be capable of effecting either constitutive or regulatedexpression of the nucleic acid molecule when transformed into a hostyeast cell. As will be readily appreciated, nucleic acid moleculesencoding one or more antigens and/or other proteins can be on one ormore expression vectors operatively linked to one or more expressioncontrol sequences. Particularly important expression control sequencesare those which control transcription initiation, such as promoter andupstream activation sequences.

Any suitable yeast promoter can be used in the present invention and avariety of such promoters are known to those skilled in the art and havegenerally be discussed above. Promoters for expression in Saccharomycescerevisiae include promoters of genes encoding the following yeastproteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1,phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI),translational elongation factor EF-1 alpha (TEF2),glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referred to asTDH3, for triose phosphate dehydrogenase), galactokinase (GAL1),galactose-1-phosphate uridyl-transferase (GAL7), UDP-galactose epimerase(GAL10), cytochrome c1 (CYC1), Sec7 protein (SECT) and acid phosphatase(PHO5), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10promoters, and including the ADH2/GAPDH promoter, which is induced whenglucose concentrations in the cell are low (e.g., about 0.1 to about 0.2percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise,a number of upstream activation sequences (UASs), also referred to asenhancers, are known. Upstream activation sequences for expression inSaccharomyces cerevisiae include the UASs of genes encoding thefollowing proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7and GAL10, as well as other UASs activated by the GAL4 gene product,with the ADH2 UAS being used in one aspect. Since the ADH2 UAS isactivated by the ADR1 gene product, it may be preferable to overexpressthe ADR1 gene when a heterologous gene is operatively linked to the ADH2UAS. Transcription termination sequences for expression in Saccharomycescerevisiae include the termination sequences of the alpha-factor, GAPDH,and CYC1 genes. Transcription control sequences to express genes inmethyltrophic yeast include the transcription control regions of thegenes encoding alcohol oxidase and formate dehydrogenase.

Likewise, transfection of a nucleic acid molecule into a yeast cellaccording to the present invention can be accomplished by any method bywhich a nucleic acid molecule administered into the cell and includesdiffusion, active transport, bath sonication, electroporation,microinjection, lipofection, adsorption, and protoplast fusion.Transfected nucleic acid molecules can be integrated into a yeastchromosome or maintained on extrachromosomal vectors using techniquesknown to those skilled in the art. As discussed above, yeast cytoplast,yeast ghost, and yeast membrane particles or cell wall preparations canalso be produced recombinantly by transfecting intact yeastmicroorganisms or yeast spheroplasts with desired nucleic acidmolecules, producing the antigen therein, and then further manipulatingthe microorganisms or spheroplasts using techniques known to thoseskilled in the art to produce cytoplast, ghost or subcellular yeastmembrane extract or fractions thereof containing desired antigens orother proteins. Further exemplary yeast expression systems, methods, andconditions suitable for use herein are described in US20100196411A1,US2017/0246276, or US 2017/0224794, and US 2012/0107347.

With respect to viral expression and vaccination systems it iscontemplated that all therapeutic recombinant viral expression systemsare deemed suitable for use herein so long as such viruses are capableto lead to expression of the recombinant payload in an infected cell.For example, suitable viruses include genetically modified alphaviruses,adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses,etc. However, adenoviruses are particularly preferred. For example,genetically modified adenoviruses are preferred that are suitable notonly for multiple vaccinations but also vaccinations in individuals withpreexisting immunity to the adenovirus (see e.g., WO 2009/006479 and WO2014/031178), typically achieved by deletion of the E2b gene and otherlate proteins to reduce immunogenicity. Moreover, due to these specificdeletions, such genetically modified viruses were replication deficientand allowed for relatively large recombinant cargo. For example, WO2014/031178 describes the use of such genetically modified viruses toexpress CEA (colorectal embryonic antigen) to provide an immune reactionagainst colon cancer. Moreover, relatively high titers of recombinantviruses can be achieved using genetically modified human 293 cells ashas been reported (e.g., J Virol. 1998 February; 72(2): 926-933).

Regardless of the type of recombinant virus it is contemplated that thevirus may be used to infect patient (or non-patient) cells ex vivo or invivo. For example, the virus may be injected subcutaneously orintravenously, or may be administered intranasaly or via inhalation toso infect the patients cells, and especially antigen presenting cells.Alternatively, immune competent cells (e.g., NK cells, T cells,macrophages, dendritic cells, etc.) of the patient (or from anallogeneic source) may be infected in vitro and then transfused to thepatient. Alternatively, immune therapy need not rely on a virus but maybe effected with nucleic acid transfection or vaccination using RNA orDNA, or other recombinant vector that leads to the expression of theneoepitopes (e.g., as single peptides, tandem mini-gene, etc.) indesired cells, and especially immune competent cells.

As noted above, the desired nucleic acid sequences (for expression fromvirus infected cells) are under the control of appropriate regulatoryelements well known in the art. For example, suitable promoter elementsinclude constitutive strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK,CAGG promoter), but inducible promoters are also deemed suitable for useherein, particularly where induction conditions are typical for a tumormicroenvironment. For example, inducible promoters include thosesensitive to hypoxia and promoters that are sensitive to TGF-β or IL-8(e.g., via TRAF, JNK, Erk, or other responsive elements promoter). Inother examples, suitable inducible promoters include thetetracycline-inducible promoter, the myxovirus resistance 1 (Mx1)promoter, etc.

Alternatively, or additionally, it should be recognized that theantigen/neoepitope or polytope may also be administered as peptide,optionally bound to a carrier protein to so act as a peptide vaccine.Among other suitable carrier proteins, human albumin or lactoferrin areparticularly preferred. Such carrier proteins may be in nativeconformation, or pretreated to form nanoparticles with exposedhydrophobic domains (see e.g., Adv Protein Chem Struct Biol. 2015;98:121-43) to which the neoepitope or polytope can be coupled. Mosttypically, coupling of the neoepitope or polytope to the carrier proteinwill be non-covalent. Similar to the secreted neoepitopes or polytopes,carrier protein-bound neoepitopes or polytopes will be taken up by theimmune competent cells, and especially antigen presenting cells anddendritic cells that in turn process and display the neoepitopes,typically via MHC-II pathways.

Formulations

Where the vaccine is a viral vaccine (e.g., an adenovirus, andespecially AdV with E1 and E2b deleted), it is contemplated that therecombinant viruses may then be individually or in combination used as atherapeutic vaccine in a pharmaceutical composition, typicallyformulated as a sterile injectable composition with a virus titer ofbetween 10⁶-10¹³ virus particles, and more typically between 10⁹-10¹²virus particles per dosage unit. Alternatively, virus may be employed toinfect patient (or other HLA matched) cells ex vivo and the so infectedcells are then transfused to the patient. In further examples, treatmentof patients with the virus may be accompanied by allografted orautologous natural killer cells or T cells in a bare form or bearingchimeric antigen receptors expressing antibodies targeting neoepitope,neoepitopes, tumor associated antigens or the same payload as the virus.The natural killer cells, which include the patient-derived NK-92 cellline, may also express CD16 and can be coupled with an antibody.

Similarly, where the vaccine is a bacterial or yeast vaccine, thebacteria or yeast cells are preferably irradiated prior toadministration to prevent further propagation. Most typically,administration is as a therapeutic vaccine in a pharmaceuticalcomposition, typically formulated as a sterile injectable compositionwith a cell titer of between 10⁶-10⁹ cells, and more typically between10⁸-10¹¹ cells per dosage unit. Most preferably, the vaccine formulationis administered intramuscularly, subcutaneously, or intratumorally. DNAvaccines will typically be administered as is well known in the art,preferably by intravenous injection of the DNA in a buffered solution asa pharmaceutical composition. While not limiting to the inventivesubject matter, the total dose of DNA per administration will typicallybe in the range of 0.1 mcg to several 10 mg, or between 10 mcg toseveral 1,000 mcg.

Where desired, additional therapeutic modalities may be employed whichmay be neoepitope based (e.g., synthetic antibodies against neoepitopesas described in WO 2016/172722), alone or in combination with autologousor allogenic NK cells, and especially haNK cells or taNK cells (e.g.,both commercially available from NantKwest, 9920 Jefferson Blvd. CulverCity, Calif. 90232). Where haNK or taNK cells are employed, it isparticularly preferred that the haNK cell carries a recombinant antibodyon the CD16 variant that binds to a neoepitope of the treated patient,and where taNK cells are employed it is preferred that the chimericantigen receptor of the taNK cell binds to a neoepitope of the treatedpatient. The additional treatment modality may also be independent ofneoepitopes, and especially preferred modalities include cell-basedtherapeutics such as activated NK cells (e.g., aNK cells, commerciallyavailable from NantKwest, 9920 Jefferson Blvd. Culver City, Calif.90232), and non cell-based therapeutics such as chemotherapy and/orradiation. In still further contemplated aspects, immune stimulatorycytokines, and especially IL-2, IL15, and IL-21 may be administered,alone or in combination with one or more checkpoint inhibitors (e.g.,ipilimumab, nivolumab, etc.). Similarly, it is still furthercontemplated that additional pharmaceutical intervention may includeadministration of one or more drugs that inhibit immune suppressivecells, and especially MDSCs Tregs, and M2 macrophages. Thus, suitabledrugs include IL-8 or interferon-γ inhibitors or antibodies binding IL-8or interferon-γ, as well as drugs that deactivate MDSCs (e.g., NOinhibitors, arginase inhibitors, ROS inhibitors), that block developmentof or differentiation of cells to MDSCs (e.g., IL-12, VEGF-inhibitors,bisphosphonates), or agents that are toxic to MDSCs (e.g., gemcitabine,cisplatin, 5-FU). Likewise, drugs like cyclophosphamide, daclizumab, andanti-GITR or anti-OX40 antibodies may be used to inhibit Tregs.

Protocols

Therefore, the inventors contemplate various exemplary strategies fortreatment with the compositions contemplated herein. Most typically,treatments will include at least two, or at least three distinct vaccinemodalities that are administered in a sequential fashion. For example insome embodiments, the initial administration will be a DNA vaccine thatis followed by administration of a bacterial vaccine. The bacterialvaccine may optionally be followed by a yeast vaccine. Following thebacterial or yeast vaccine administration may then be a viral vaccineadministration. In another example, the initial administration will be abacterial vaccine that is optionally followed by administration of ayeast vaccine. Following the bacterial or yeast vaccine administrationmay then be a viral vaccine administration. In still another example,the initial administration will be a DNA vaccine, which is followed by aviral vaccine administration. As will be readily appreciated, each ofthe modalities can be administered once, or repeatedly as desired. Forexample, the DNA vaccine may be given once, while a subsequent bacterialand/or yeast vaccine may be administered twice, three times or more,before administration of a viral vaccine commences. Similarly, multiplebacterial or yeast vaccine compositions may be administered prior to aviral vaccine composition. In the same way, the DNA, bacterial, and/oryeast vaccine may be given once and the viral vaccine may be givenmultiple times. Therefore, it should be noted that in a prime/boostregimen, the prime vaccination may use a modality that is different fromthe boost vaccination (e.g., DNA, bacterial, or yeast vaccination asprime, viral vaccination as boost).

It should further be recognized that the administrations of the specificmodalities will be spaced apart by several days to allow an immuneresponse to develop. Most typically, the first administration will bespaced apart from the second administration by at least two days, moretypically by at least four days, even more typically by at least oneweek, and most typically by two weeks or even longer. Moreover, itshould be noted that the immune system of the patient may also bepre-conditioned using immune stimulatory cytokines (e.g., IL-2, IL-15,IL-21) or cytokine analogs (e.g., ALT-803), using checkpoint inhibitorsor Treg or M2 macrophage inhibitors to reduce immune suppression, orusing cytokines that support an immune response and generation of immunememory (e.g., IL-12).

As used herein, the term “administering” a pharmaceutical composition ordrug refers to both direct and indirect administration of thepharmaceutical composition or drug, wherein direct administration of thepharmaceutical composition or drug is typically performed by a healthcare professional (e.g., physician, nurse, etc.), and wherein indirectadministration includes a step of providing or making available thepharmaceutical composition or drug to the health care professional fordirect administration (e.g., via injection, infusion, oral delivery,topical delivery, etc.). Most preferably, the recombinant virus isadministered via subcutaneous or subdermal injection. However, in othercontemplated aspects, administration may also be intravenous injection.Alternatively, or additionally, antigen presenting cells may be isolatedor grown from cells of the patient, infected in vitro, and thentransfused to the patient. Therefore, it should be appreciated thatcontemplated systems and methods can be considered a complete drugdiscovery system (e.g., drug discovery, treatment protocol, validation,etc.) for highly personalized cancer treatment. As will also beappreciated, contemplated treatments may be repeated over time,particularly where new neoepitopes have developed (e.g., as a result ofclonal expansion).

Moreover, it is noted that additional treatment regimens may beimplemented to assist contemplated methods and compositions. Suchadditional treatment regimens will preferably be performed to increase‘visibility’ of a tumor to the immune system. For example, to triggeroverexpression or transcription of stress signals, it is contemplatedthat chemotherapy and/or radiation may be employed using at a low-doseregimen, preferably in a metronomic fashion. For example, it isgenerally preferred that such treatment will use doses effective toaffect at least one of protein expression, cell division, and cellcycle, preferably to induce apoptosis or at least to induce or increasethe expression of stress-related genes (and particularly NKG2D ligands).Thus, in further contemplated aspects, such treatment will include lowdose treatment using one or more chemotherapeutic agents. Mosttypically, low dose treatments will be at exposures that are equal orless than 70%, equal or less than 50%, equal or less than 40%, equal orless than 30%, equal or less than 20%, equal or less than 10%, or equalor less than 5% of the LD₅₀ or IC₅₀ for the chemotherapeutic agent.Additionally, where advantageous, such low-dose regimen may be performedin a metronomic manner as described, for example, in U.S. Pat. Nos.7,758,891, 7,771,751, 7,780,984, 7,981,445, and 8,034,375.

With respect to the particular drug used in low-dose regimens, it iscontemplated that all chemotherapeutic agents are deemed suitable. Amongother suitable drugs, kinase inhibitors, receptor agonists andantagonists, anti-metabolic, cytostatic and cytotoxic drugs are allcontemplated herein. However, particularly preferred agents includethose identified to interfere or inhibit a component of a pathway thatdrives growth or development of the tumor. Suitable drugs can beidentified using pathway analysis on omics data as described in, forexample, WO 2011/139345 and WO 2013/062505. Most notably, so achievedexpression of stress-related genes in the tumor cells will result insurface presentation of NKG2D, NKP30, NKP44, and/or NKP46 ligands, whichin turn activate NK cells to specifically destroy the tumor cells. Thus,it should be appreciated that low-dose chemotherapy may be employed as atrigger in tumor cells to express and display stress related proteins,which in turn will trigger NK-cell activation and/or NK-cell mediatedtumor cell killing. Additionally, NK-cell mediated killing will beassociated with release of intracellular tumor specific antigens, whichis thought to further enhance the immune response.

EXAMPLES

Exemplary Sequence Arrangements

Neoepitope sequences were determined in silico by location-guidedsynchronous alignment of tumor and normal samples as, for example,disclosed in US 2012/0059670 and US 2012/0066001 using BAM files and BAMservers. Specifically, DNA analysis of the tumor was from the B16-F10mouse melanoma line and matched normal was blood from C57bl/6 parentalmouse DNA. The results were filtered for expression by RNA sequencing ofthis tumor cell line. Neoepitopes that were found expressed were furtheranalyzed for binding affinity towards murine MHC-I (here: Kb) and MHC-II(here: I-Ab). Selected binders (with affinity of equal or less than 200nM) were further analyzed after a further step of dbSNP filtering usingpositional permutations of all neoepitopes that were then processedthrough a weight matrix and neural network prediction to generate ascore representing the likelihood of presence and/or strength ofhydrophobic sequences or signal peptides. The best scoring arrangement(lowest likelihood of hydrophobic sequences or signal peptides) for thepolytope (not shown) was used for further experiments. Neoepitopes wereprioritized by detection in RNAseq or other quantitative system thatyielded expression strength for a specific gene harboring the neoepitopemutation.

Table 1 shows exemplary neoepitopes that were expressed as determined byRNAseq along with gene name and mutated amino acid and position of themutated amino acid. The neoepitope listed with * was discarded afterdbSNP filtering as that neoepitope occurred as variant Rs71257443 in 28%of the population.

TABLE 1 Gene Position Neoepitope-a Neoepitope-b VIPR2 V73M GETVTMPCPLILRB3 T187N VGPVNPSHR* FCRL1 R286C GLGAQCSEA FAT4 S1613L RKLTTELTIPERRKLTTE PIEZO2 T2356M MDWVWMDTT VWMDTTLSL SIGLEC14 A292T GKTLNPSQTREGKTLNPS SIGLEC1 D1143N VRNATSYRC NVTVRNATS SLC4A11 Q678P FAMAQIPSLAQIPSLSLR

Table 2 shows further examples of neoepitopes in which the position ofthe mutated amino acid was changed, and shows further alternatesequences for MHC-I presentation (9-mer) and MHC-II presentation(15-mer). The neoepitope sequence for MHC-II presentation wasback-translated to the corresponding nucleic acid sequence, which isalso shown in Table 2.

TABLE 2 Nucleotide  Gene Change Neoepitope-a Neoepitope-bExtended 15 mer Sequence SLC4A11 Q678P FAMAQIPSL AQIPSLSLRPFAMAQIPSLSLRAV CCCTTCGCCATGGCC CAGATCCCCAGCCTG AGCCTGAGGGCCGTG SIGLEC1D1143N VRNATSYRC NVTVRNATS LPNVTVRNATSYRCG CTGCCCAACGTGACCGTGAGGAACGCCACC AGCTACAGGTGCGGC SIGLEC14 A292T GKTLNPSQT REGKTLNPSSWFREGKTLNPSQTS AGCTGGTTCAGGGAG GGCAAGACCCTGAAC CCCAGCCAGACCAGC PIEZO2T2356M MDWVWMDTT VWMDTTLSL AVMDWVWMDTTLSLS GCCGTGATGGACTGGGTGTGGATGGACACC ACCCTGAGCCTGAGC FAT4 S1613L RKLTTELTI PERRKLTTELGPERRKLTTELTII CTGGGCCCCGAGAGG AGGAAGCTGACCACC GAGCTGACCATCATC FCRL1R286C GLGAQCSEA NNGLGAQCSEAVTLN AACAACGGCCTGGGC GCCCAGTGCAGCGAGGCCGTGACCCTGAAC VIPR2 V73M GETVTMPCP NVGETVTMPCPKVFS AACGTGGGCGAGACCGTGACCATGCCCTGC CCCAAGGTGTTCAGC FLRT2 R346W EQVWGMAVR CQGPEQVWGMAVRELTGCCAGGGCCCCGAG CAGGTGTGGGGCATG GCCGTGAGGGAGCTG

Sequence Trafficking

Model cancer: Murine B16-F10 melanoma (derived from C57/B16 mouse) wasused tumors were screened in a tumor versus normal manner as describedabove, and expressed mutant epitopes were identified in the B16F10melanoma cell line. Candidate neoepitopes were further filtered asdescribed above using sequencing data analysis and binding analysis tomurine MHC I (H2-Kb, H2-Dd) and MHC II (I-Ab). Nine distinct polytopeconstructs were then prepared for testing various trafficking schemes,and each construct was prepared as the corresponding recombinant nucleicacid under the control of a CMV promoter. Each construct was cloned intoan AdV5 expression vector that had deleted E1 and E2b genes, and theresulting recombinant virus was then used for transfection of mice as isfurther discussed below.

More specifically, three polytope constructs included MHC I bindingneoepitopes for MHC-I presentation and were therefore targeted to thecytoplasmic compartment. While one construct had an unmodifiedN-terminus, another construct had an N-terminal non-cleavable ubiquitin,and yet another construct had an N-terminal cleavable ubiquitin.Ubiquitination was used to target the proteasome in the cytosol. Threefurther polytope constructs included MHC I binding neoepitopes forMHC-II presentation and were therefore targeted to thelysosomal/endosomal compartments compartment. While one construct hadlysosomal targeting sequence, another construct had a recyclingendosomal targeting sequence, and yet another construct had a sortingendosomal targeting sequence. Three additional polytope constructsincluded MHC II binding neoepitopes for MHC-II presentation and werealso targeted to the lysosomal/endosomal compartments compartment. Oncemore, one construct had lysosomal targeting sequence, another constructhad a recycling endosomal targeting sequence, and yet another constructhad a sorting endosomal targeting sequence. These nine constructs hadsequence arrangements as follows.

In the following exemplary sequences, for MHC-I presentation, ubiquitin(cleavable and non-cleavable) were used for proteasome targeting, whilethe CD1b leader peptide was used as an export leader peptide fortrafficking the polypeptide out of the cytosol for all MHC-II directedsequences. LAMP1-TM/cytoplasmic tail was used as a lysosomal targetingsequence, while LAMP1-TM/CD1a tail was used as a recycling endosomestargeting sequence, and LAMP1-TM/CD1c tail was used as a sortingendosomes targeting domain.

It should further be noted that various internal controls were also usedin the above polypeptides to account for expression and presentation.More specifically, the SIINFEKL peptide was used as an internal controlfor a MHC I restricted (Kb) peptide epitope, while the Esat6 peptide wasused as an internal control for a secreted protein for MHC IIpresentation. FLAG-tag was used as an internal control epitope fordetection of expression, and cMYC used as an internal control Tag forsimple protein detection.

Exemplary Constructs for MHC-I Epitopes Directed to MHC-I Presentation(Traffic Through Proteasome, Cytoplasmic Targeting)

Polyepitope only:12aa-A^(m)-12aa-AAAA-12aa-B^(m)-12aa-AAAA-(12aa-X^(m)-12aa-AAAA)_(n)-SIINFEKL-AAAA-Esat6-cMYC.FIG. 5A exemplarily depicts the polypeptide structure of sucharrangement where the SIINFEKL motif is underlined, the Esat6 motif isin italics, and where the cMY motif is in bold type font. The nucleotidesequence for FIG. 5A is SEQ ID NO: 1, and the polypeptide sequence forFIG. 5A is SEQ ID NO: 4.

Polyepitope and cleavable ubiquitin GGR N-terminus:Ubiquitin-GGR-12aa-A^(m)-12aa-AAAA-12aa-B^(m)-12aa-AAAA-(12aa-X^(m)-12aa-AAAA)_(n)-SIINFEKL-AAAA-Esat6-cMYC.FIG. 5B exemplarily depicts the polypeptide structure of sucharrangement where the cleavable ubiquitin moiety is italics andunderlined, the SIINFEKL motif is underlined, the Esat6 motif is initalics, and where the cMY motif is in bold type font. The nucleotidesequence for FIG. 5B is SEQ ID NO: 2, and the polypeptide sequence forFIG. 5B is SEQ ID NO: 5.

Polyepitope and non-cleavable ubiquitin G N-terminus:Ubiquitin-G-12aa-A^(m)-12aa-AAAA-12aa-B^(m)-12aa-AAAA-(12aa-X^(m)-12aa-AAAA)_(n)-SIINFEKL-AAAA-Esat6-cMYC.FIG. 5C exemplarily depicts the polypeptide structure of sucharrangement where the cleavable ubiquitin moiety is italics andunderlined, the SIINFEKL motif is underlined, the Esat6 motif is initalics, and where the cMY motif is in bold type font. The nucleotidesequence for FIG. 5C is SEQ ID NO: 3, and the polypeptide sequence forFIG. 5C is SEQ ID NO: 6.

Exemplary Constructs for MHC-I Epitopes Directed to MHC-II Presentation(Export from Cytoplasm, Traffic Through Endo/Lysosome)

Lysosomal targeting of Kb epitope peptides: (CD1b leaderpeptide)-20aa-A^(m)-20aa-GPGPG-20aaB^(m)-20aa-GPGPG-(20aa-X^(m)-20aa-GPGPG-)_(n)-Esat6-FlagTag-LAMP1TM/cytoplasmictail. FIG. 6A exemplarily depicts the polypeptide structure of sucharrangement where the CD1b leader peptide moiety is bold, the Esat6motif is in underlined, the Flag-tag motif is italics, and where theLAMP1TM/cytoplasmic tail is in bold/underline type font. The nucleotidesequence for FIG. 6A is SEQ ID NO: 7, and the polypeptide sequence forFIG. 6A is SEQ ID NO: 10.

Recycling lysosome targeting of Kb epitope peptides: (CD1b leaderpeptide)-20aa-A^(m)-20aa-GPGPG-20aaB^(m)-20aa-GPGPG-(20aa-X^(m)-20aa-GPGPG-)_(n)-Esat6-FlagTag-LAMP1TM/CD1atail. FIG. 6B exemplarily depicts the polypeptide structure of sucharrangement where the CD1b leader peptide moiety is bold, the Esat6motif is in underlined, the Flag-tag motif is italics, and where theLAMP1TM motif is in bold/underline type font, and the CD1a targetingmotif is underline/italics. The nucleotide sequence for FIG. 6B is SEQID NO: 8, and the polypeptide sequence for FIG. 6B is SEQ ID NO: 11.

Sorting endosome targeting of Kb epitope peptides: (CD1b leaderpeptide)-20aa-A^(m)-20aa-GPGPG-20aaB^(m)-20aa-GPGPG-(20aa-X^(m)-20aa-GPGPG-)_(n)-Esat6-FlagTag-LAMP1TM/CD1ctail. FIG. 6C exemplarily depicts the polypeptide structure of sucharrangement where the CD1b leader peptide moiety is bold, the Esat6motif is in underlined, the Flag-tag motif is italics, and where theLAMP1TM motif is in bold/underline type font, and the CD1c targetingmotif is underline/italics. The nucleotide sequence for FIG. 6C is SEQID NO: 9, and the polypeptide sequence for FIG. 6C is SEQ ID NO: 12.

Exemplary Constructs for MHC-II Epitopes Directed to MHC-II Presentation(Export from Cytoplasm, Traffic Through Endo/Lysosome)

Lysosomal targeting of IAb epitope peptides: (CD1b leaderpeptide)-20aa-A^(m)-20aa-GPGPG-20aaB^(m)-20aa-GPGPG-(20aa-X^(m)-20aa-GPGPG-)_(n)-Esat6-FlagTag-LAMP1TM/cytoplasmictail. FIG. 7A exemplarily depicts the polypeptide structure of sucharrangement where the CD1b leader peptide moiety is bold, the SIINFEKLand Esat6 motifs are underlined, the Flag-tag motif is italics, andwhere the LAMP1TM/cytoplasmic tail is in bold/underline type font. Thenucleotide sequence for FIG. 7A is SEQ ID NO: 13, and the polypeptidesequence for FIG. 7A is SEQ ID NO: 16.

Recycling lysosome targeting of IAb epitope peptides: (CD1b leaderpeptide)-20aa-A^(m)-20aa-GPGPG-20aaB^(m)-20aa-GPGPG-(20aa-X^(m)-20aa-GPGPG-)_(n)-Esat6-FlagTag-LAMP1TM/CD1atail. FIG. 7B exemplarily depicts the polypeptide structure of sucharrangement where the CD1b leader peptide moiety is bold, the SIINFEKLand Esat6 motifs are underlined, the Flag-tag motif is italics, wherethe LAMP1TM motif is in bold/underline type font, and where the CD1atail is in bold/italics. The nucleotide sequence for FIG. 7B is SEQ IDNO: 14, and the polypeptide sequence for FIG. 7B is SEQ ID NO: 17.

Sorting endosome targeting of IAb epitope peptides: (CD1b leaderpeptide)-20aa-A^(m)-20aa-GPGPG-20aaB^(m)-20aa-GPGPG-(20aa-X^(m)-20aa-GPGPG-)_(n)-Esat6-FlagTag-LAMP1TM/CD1ctail. FIG. 7C exemplarily depicts the polypeptide structure of sucharrangement where the CD1b leader peptide moiety is bold, the SIINFEKLand Esat6 motifs are underlined, the Flag-tag motif is italics, wherethe LAMP1TM motif is in bold/underline type font, and where the CD1ctail is in bold/italics. The nucleotide sequence for FIG. 7C is SEQ IDNO: 15, and the polypeptide sequence for FIG. 7C is SEQ ID NO: 18.

In Vivo Vaccination

FIG. 8 depicts an exemplary in vivo vaccination experiment where ninegroups of C57bl/6 mice were immunized with nine distinct recombinant Ad5viruses comprising the sequence arrangements substantially as describedabove. Immunization followed a biweekly schedule of administration withdistinct routes for separate groups of animals (subcutaneous andintravenous) as is schematically shown in FIG. 8. Tumor challenge withB16-F10 melanoma cells was at day 42, followed by administration of anM2 macrophage suppressive drug (RP 182) and IL-15 superagonist(Alt-803). FIGS. 9A-9C depict exemplary results for subcutaneousadministration, while FIGS. 10A-10C depict exemplary results forintravenous administration.

Notably, subcutaneous injection of adenovirus encoding Class I polytopesdirected to the cytoplasm and MHC-I presentation did not provide asignificant immune protection, regardless of the presence or absence ofubiquitination as can be taken from FIG. 9A. On the other hand, wherethe Class I polytopes were directed to the endosomal and lysosomalcompartments for processing and presentation via MHC-II, some protectiveimmunity was observed for direction to the recycling endosomalcompartment and lysosomal compartment as is evident from FIG. 9B. Evenstronger immune protection was observed when Class II polytopes weredirected to the endosomal and lysosomal compartments for processing andpresentation via MHC-II. Here, the strongest protection was observed forlysosomal and sorting endosomal compartments as is shown in FIG. 9C.

When immunization was performed with the same viral constructs, albeitvia intravenous injection, protective effect of neoepitope vaccinationwas observed for Class I neoepitopes directed to the cytoplasm where thepolytope included cleavable ubiquitin, and some protective effect wasobserved where the polytope included non-cleavable ubiquitin as can beseen from FIG. 10A. Notably, when the Class I polytopes were directed tothe endosomal and lysosomal compartment, stronger protective effect wasobserved in all vaccinations as is shown in FIG. 10B. Moreover, strongprotective effect was observed when Class II polytopes were directed tothe endosomal and lysosomal compartments for processing and presentationvia MHC-II. Here, the strongest protection was observed for recyclingand sorting endosomal compartments as is shown in the graph of FIG. 10C.

Comparison of Routes and Vectors

In still further experiments, the inventors further investigated if theactual route of administration and type of expression vector had aneffect on the therapeutic efficacy. To that end, a substantiallyidentical mouse model as described above was employed and the polytopewas constructed from neoepitopes from B16F10 melanoma cells. FIGS. 11Aand 11B show exemplary results for the experiments where the expressionvector was an adenovirus. As can be readily seen, subcutaneousadministration of an adenoviral expression system encoding MHC IItargeted polytope conferred a significant immune protective effect forall sub-cellular locations while a null vector failed to provide immuneprotective effect as can be taken from FIG. 11A. Notably, when the samevector constructs were tested using intravenous administration, immuneprotective effect was less pronounced as can be taken from FIG. 11B.

Conversely, where the expression vector was a plasmid (here: shuttlevector for generating the adenoviral expression vector) targeting MHC IIpresentation as above, subcutaneous administration of the plasmidconferred a notable immune protective effect versus null vector as isshown in FIG. 11C. Moreover, and unexpectedly, where the same plasmidwas administered by intravenous injection, a substantial immuneprotective effect was observed, even for the null vector as can be takenfrom FIG. 11D. While not wishing to be bound by any particular theory orhypothesis, the inventors therefore contemplate that the immuneprotective effect may be at least in part due to an innate and anadaptive immune response.

FIG. 11E illustrates a comparison between use of different routes usingempty plasmid (‘null’) versus empty viral (‘null’) expression vector. Ascan be readily seen from the results, subcutaneous administration didnot confer immune protective effect, while the strongest immuneprotective effect was observed by intravenous administration of nullplasmid.

FIG. 12 depicts the data shown in Table 3 below where the type ofexpression vector is shown as “Type”, and the route of administrationindicated as “Route”. The particular MHC targeting and targeting ofsub-cellular location is shown under “Nucleic Acid”, while tumor volumeis indicated for the dates measured after implantation of B16-F10melanoma cells.

TABLE 3 # Type Route Nucleic Acid Day 7 Day 10 Day 14 Day 17 Day 21 Day24 1 AdV Sub Q Null AdV 29.49791667 57.92242 260.014833 688.76291246.774 2119.323 2 AdV Sub Q K^(b)-Cyto AdV 23.97941667 39.50383187.24075 529.0868 1715.039 2993.372 3 AdV Sub Q K^(b)-UBQ (cleavable)AdV 26.4185 50.95125 253.255 685.7033 1882.06 3773.93 4 AdV Sub QI-A^(b)-CD1a AdV 16.135 16.195 26.6793333 60.27433 157.3007 341.9303 5AdV Sub Q I-A^(b)-CD1c AdV 15.75 17.37667 29.32725 59.65917 257.8108385.198 6 AdV Sub Q I-A^(b)-LAMP1 AdV 17.64666667 18.82333 21.927550.01192 116.2528 310.7113 7 AdV IV Null AdV 29.06025 60.5985 219.399833531.3525 990.4138 2527.871 8 AdV IV K^(b)-Cyto AdV 36.07041667 48.72017113.244 408.8533 1372.974 2887.699 9 AdV IV K^(b)-UBQ (cleavable) AdV26.62333333 65.407 202.246667 438.495 1565.227 3128.09 10 AdV IVI-A^(b)-CD1a AdV 22.08333333 44.61333 114.910333 267.6967 767.71631018.658 11 AdV IV I-A^(b)-CD1c AdV 28.1535 34.5925 62.64375 169.7857542.2878 1140.391 12 AdV IV I-A^(b)-LAMP1 AdV 29.82733333 70.32883183.320333 344.7302 648.9571 1033.862 13 Plasmid Sub Q Null/pShuttleplasmid 38.22775 76.19925 263.548875 620.3451 837.5773 1473.021 14Plasmid Sub Q K^(b)-Cyto Plasmid 31.7225 81.33738 277.45375 724.64571107.705 2506.988 15 Plasmid Sub Q K^(b)-UBQ (cleavable) 19.42 40.41075181.246 454.0527 951.4715 1995.16 Plasmid 16 Plasmid Sub Q I-A^(b)-CD1aPlasmid 25.595 32.52 70.540625 179.315 522.0373 1340.285 17 Plasmid SubQ I-A^(b)-CD1c Plasmid 15.19 28.58125 149.4485 358.5129 933.29261681.392 18 Plasmid Sub Q I-A^(b)-LAMP1 Plasmid 19.6025 32.6962580.381875 160.4238 542.5311 1493.744 19 Plasmid IV Null/pShuttle Plasmid21.3825 42.272 116.60725 324.2005 659.2577 1528.49 20 Plasmid IVK^(b)-Cyto Plasmid 14.155 30.05125 106.206 319.6288 562.9468 1113.58 21Plasmid IV K^(b)-UBQ (cleavable) 20.4275 45.167 198.383625 486.28561111.359 2619.425 Plasmid 22 Plasmid IV I-A^(b)-CD1a Plasmid 16.4522.293 61.65325 151.3369 554.821 1612.671 23 Plasmid IV I-A^(b)-CD1cPlasmid 11.725 13.7225 27.265125 73.65725 184.9038 447.9333 24 PlasmidIV I-A^(b)-LAMP1 Plasmid 15.6875 19.625 70.805875 259.7303 422.6391223.331

As can be readily seen from the data presented here, targeting MHC-IImatched neoepitopes of a polytope and targeting the polytope towardsMHC-II presentation via CD1c, LAMP1, and CD1a was significantlyeffective when administered intravenously in plasmid form, andsubcutaneously in adenovirus form. Notably, and as also reflected in thedata, targeting MHC-I was significantly less effective to provide immuneprotection.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of generating recombinant expressionconstructs for use in immune therapy in a mammal, comprising: generatinga first recombinant nucleic acid having a sequence that encodes apolytope, wherein the polytope comprises a plurality of filteredneoepitope sequences; wherein the polytope further comprises atrafficking element that directs the polytope to a sub-cellular locationselected from the group consisting of a recycling endosome, a sortingendosome, and a lysosome; wherein the first recombinant nucleic acidcomprises a first promoter operably linked to the sequence that encodesthe polytope to drive expression of the polytope in the mammal; andgenerating a second recombinant nucleic acid having the sequence thatencodes the polytope, wherein the second recombinant nucleic acidcomprises a second promoter operably linked to the sequence that encodesthe polytope to drive expression of the polytope in a non-mammaliancell.
 2. The method of claim 1 wherein the first promoter is aconstitutive promoter or wherein the first promoter is inducible byhypoxia, IFN-gamma, or IL-8.
 3. The method of any one of the precedingclaims wherein the trafficking element is selected from the groupconsisting of a CD1b leader sequence, a CD1a tail, a CD1c tail, and aLAMP1-transmembrane sequence.
 4. The method of any one of the precedingclaims wherein the filtered neoepitope sequences are filtered bycomparing tumor versus matched normal of the same patient.
 5. The methodof any one of the preceding claims wherein the filtered neoepitopesequences are filtered to have binding affinity to an MHC complex ofequal or less than 200 nM.
 6. The method of any one of claims 1-5wherein the filtered neoepitope sequences bind to MHC-I and wherein thetrafficking element directs the polytope to the recycling endosome,sorting endosome, or lysosome.
 7. The method of any one of claims 1-5wherein the filtered neoepitope sequences bind to MHC-II and wherein thetrafficking element directs the polytope to the recycling endosome,sorting endosome, or lysosome.
 8. The method of any one of the precedingclaims wherein the first recombinant nucleic acid further comprises anadditional sequence that encodes a second polytope, wherein the secondpolytope comprises a second trafficking element that directs the secondpolytope to a different sub-cellular location and wherein the secondpolytope comprises a second plurality of filtered neoepitope sequences.9. The method of claim 8 wherein at least one of the filtered neoepitopesequences and at least one of the second filtered neoepitope sequencesare identical.
 10. The method of any one of the preceding claims whereinthe first recombinant nucleic acid further comprises a sequence thatencodes at least one of a co-stimulatory molecule, an immune stimulatorycytokine, and a protein that interferes with or down-regulatescheckpoint inhibition.
 11. The method of claim 10 wherein theco-stimulatory molecule is selected from the group consisting of CD80,CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L,4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3. 12.The method of claim 10 wherein the immune stimulatory cytokine isselected from the group consisting of IL-2, IL-12, IL-15, IL-15 superagonist (ALT803), IL-21, IPS1, and LMP1.
 13. The method of claim 10wherein the protein that interferes is an antibody or an antagonist ofCTLA-4, PD-1, TIM1 receptor, 2B4, or CD160.
 14. The method of any one ofthe preceding claims wherein the first recombinant nucleic acid isreplicated in a bacterial cell or a yeast cell.
 15. The method of anyone of the preceding claims wherein the first recombinant nucleic acidis a shuttle vector for generation of a recombinant virus.
 16. Themethod of claim 15 wherein the recombinant virus is an adenovirus,optionally with at least one of an E1 and an E2b gene deleted.
 17. Themethod of any one of the preceding claims further comprising a step offormulating the first recombinant nucleic acid into a pharmaceuticalformulation for injection.
 18. The method of any one of the precedingclaims wherein the second promoter is a constitutive bacterial or ayeast promoter.
 19. The method of any one of the preceding claimswherein the non-mammalian cell is an E. coli cell or a Saccharomycescerevisiae cell.
 20. The method of any one of the preceding claimsfurther comprising the steps of: transfecting the second recombinantnucleic acid into a bacterial cell or a yeast cell; expressing thepolytope in the bacterial cell or the yeast cell; and formulating thebacterial cell or the yeast cell into a pharmaceutical formulation forinjection.
 21. The method of claim 1 wherein the trafficking element isselected from the group consisting of a CD1b leader sequence, a CD1atail, a CD1c tail, and a LAMP1-transmembrane sequence.
 22. The method ofclaim 1 wherein the filtered neoepitope sequences are filtered bycomparing tumor versus matched normal of the same patient.
 23. Themethod of claim 1 wherein the filtered neoepitope sequences are filteredto have binding affinity to an MHC complex of equal or less than 200 nM.24. The method of claim 1 wherein the filtered neoepitope sequences bindto MHC-I and wherein the trafficking element directs the polytope to therecycling endosome, sorting endosome, or lysosome.
 25. The method ofclaim 1 wherein the filtered neoepitope sequences bind to MHC-II andwherein the trafficking element directs the polytope to the recyclingendosome, sorting endosome, or lysosome.
 26. The method of claim 1wherein the first recombinant nucleic acid further comprises anadditional sequence that encodes a second polytope, wherein the secondpolytope comprises a second trafficking element that directs the secondpolytope to a different sub-cellular location and wherein the secondpolytope comprises a second plurality of filtered neoepitope sequences.27. The method of claim 26 wherein at least one of the filteredneoepitope sequences and at least one of the second filtered neoepitopesequences are identical.
 28. The method of claim 1 wherein the firstrecombinant nucleic acid further comprises a sequence that encodes atleast one of a co-stimulatory molecule, an immune stimulatory cytokine,and a protein that interferes with or down-regulates checkpointinhibition.
 29. The method of claim 28 wherein the co-stimulatorymolecule is selected from the group consisting of CD80, CD86, CD30,CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L,TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3.
 30. The method ofclaim 28 wherein the immune stimulatory cytokine is selected from thegroup consisting of IL-2, IL-12, IL-15, IL-15 super agonist (ALT803),IL-21, IPS1, and LMP1.
 31. The method of claim 28 wherein the proteinthat interferes is an antibody or an antagonist of CTLA-4, PD-1, TIM1receptor, 2B4, or CD160.
 32. The method of claim 1 wherein the firstrecombinant nucleic acid is replicated in a bacterial cell or a yeastcell.
 33. The method of claim 1 wherein the first recombinant nucleicacid is a shuttle vector for generation of a recombinant virus.
 34. Themethod of claim 33 wherein the recombinant virus is an adenovirus,optionally with at least one of an E1 and an E2b gene deleted.
 35. Themethod of claim 1 further comprising a step of formulating the firstrecombinant nucleic acid into a pharmaceutical formulation forinjection.
 36. The method of claim 1 wherein the second promoter is aconstitutive bacterial or a yeast promoter.
 37. The method of claim 1wherein the non-mammalian cell is an E. coli cell or a Saccharomycescerevisiae cell.
 38. The method of claim 1 further comprising the stepsof: transfecting the second recombinant nucleic acid into a bacterialcell or a yeast cell; expressing the polytope in the bacterial cell orthe yeast cell; and formulating the bacterial cell or the yeast cellinto a pharmaceutical formulation for injection.
 39. A recombinantbacterial or yeast expression vector for immune therapy of a mammal,comprising: a recombinant nucleic acid having a sequence that encodes apolytope operably linked to a bacterial or yeast promoter to driveexpression of the polytope; wherein the polytope comprises a traffickingelement that directs the polytope to a sub-cellular location of amammalian immune competent cell selected from the group consisting ofrecycling endosome, sorting endosome, and lysosome; and wherein thepolytope comprises a plurality of filtered neoepitope sequences.
 40. Thevector of claim 39 wherein the promoter is a constitutive promoter. 41.The vector of any one of claims 39-40 wherein the trafficking element isselected from the group consisting of a CD1b leader sequence, a CD1atail, a CD1c tail, and a LAMP1-transmembrane sequence.
 42. The vector ofany one of claims 39-41 wherein the filtered neoepitope sequences arefiltered by comparing tumor versus matched normal of the same patient.43. The vector of any one of claims 39-42 wherein the filteredneoepitope sequences bind to MHC-I and wherein the trafficking elementdirects the polytope to the recycling endosome, sorting endosome, orlysosome.
 44. The vector of any one of claims 39-42 wherein the filteredneoepitope sequences bind to MHC-II and wherein the trafficking elementdirects the polytope to the recycling endosome, sorting endosome, orlysosome.
 45. The vector of any one of claims 39-45 wherein therecombinant nucleic acid further comprises an additional sequence thatencodes a second polytope, wherein the second polytope comprises asecond trafficking element that directs the second polytope to adifferent sub-cellular location and wherein the second polytopecomprises a second plurality of filtered neoepitope sequences.
 46. Thevector of claim 45 wherein at least one of the filtered neoepitopesequences and at least one of the second filtered neoepitope sequencesare identical.
 47. The vector of any one of claims 39-46 wherein theexpression vector is a bacterial expression vector.
 48. The vector ofany one of claims 39-46 wherein the expression vector is a yeastexpression vector.
 49. The vector of claim 39 wherein the traffickingelement is selected from the group consisting of a CD1b leader sequence,a CD1a tail, a CD1c tail, and a LAMP1-transmembrane sequence.
 50. Thevector of claim 39 wherein the filtered neoepitope sequences arefiltered by comparing tumor versus matched normal of the same patient.51. The vector of claim 39 wherein the filtered neoepitope sequencesbind to MHC-I and wherein the trafficking element directs the polytopeto the recycling endosome, sorting endosome, or lysosome.
 52. The vectorof claim 39 wherein the filtered neoepitope sequences bind to MHC-II andwherein the trafficking element directs the polytope to the recyclingendosome, sorting endosome, or lysosome.
 53. The vector of claim 39wherein the recombinant nucleic acid further comprises an additionalsequence that encodes a second polytope, wherein the second polytopecomprises a second trafficking element that directs the second polytopeto a different sub-cellular location and wherein the second polytopecomprises a second plurality of filtered neoepitope sequences.
 54. Thevector of claim 53 wherein at least one of the filtered neoepitopesequences and at least one of the second filtered neoepitope sequencesare identical.
 55. The vector of claim 39 wherein the expression vectoris a bacterial expression vector.
 56. The vector of claim 39 wherein theexpression vector is a yeast expression vector.
 57. A recombinant yeastcell transfected with the vector of any one of claims 40-48.
 58. Arecombinant yeast cell transfected with the vector of any one of claims49-56.
 59. A recombinant bacterial cell transfected with the vector ofany one of claims 40-48.
 60. A recombinant bacterial cell transfectedwith the vector of any one of claims 49-56.
 61. A pharmaceuticalcomposition comprising the recombinant yeast cell of claim
 57. 62. Apharmaceutical composition comprising the recombinant yeast cell ofclaim
 58. 63. A pharmaceutical composition comprising the recombinantbacterial cell of claim
 59. 64. A pharmaceutical composition comprisingthe recombinant bacterial cell of claim
 60. 65. (DNA/cell-based) Amethod of preparing first and second treatment compositions for anindividual having a tumor, comprising: identifying a plurality ofexpressed neoepitope sequences from omics data of the tumor, whereineach of the expressed neoepitope sequences have a calculated bindingaffinity of equal or less than 500 nM to at least one of MHC-I andMHC-II of the individual; generating a first recombinant nucleic acidhaving a sequence that encodes a polytope, wherein the polytopecomprises the plurality of expressed neoepitope sequences; wherein thepolytope further comprises a trafficking element that directs thepolytope to a sub-cellular location selected from the group consistingof a recycling endosome, a sorting endosome, and a lysosome; wherein thefirst recombinant nucleic acid comprises a first promoter operablylinked to the sequence that encodes the polytope to drive expression ofthe polytope in a cell of the individual; formulating the firstrecombinant nucleic into a DNA vaccine formulation to so obtain thefirst treatment composition; generating a second recombinant nucleicacid that includes the sequence that encodes the polytope, wherein thesecond recombinant nucleic acid comprises a second promoter operablylinked to the sequence that encodes the polytope to drive expression ofthe polytope in a bacterial cell or a yeast cell; transfecting thebacterial cell or the yeast cell with the second recombinant nucleicacid and expressing the polytope in the bacterial cell or the yeastcell; and formulating the transfected bacterial cell or the yeast cellinto a cell-based vaccine formulation to so obtain the second treatmentcomposition.
 66. The method of claim 65 wherein the plurality ofexpressed neoepitope sequences are identified using incrementalsynchronous alignment of omics data from the tumor and omics data from anon-tumor sample of the same individual.
 67. The method of any one ofclaims 65-66 wherein the first recombinant nucleic acid is an expressionvector.
 68. The method of any one of claims 65-67 wherein thetrafficking element is selected from the group consisting of a CD1bleader sequence, a CD1a tail, a CD1c tail, and a LAMP1-transmembranesequence.
 69. The method of any one of claims 65-68 wherein the secondpromoter is a constitutive bacterial or a yeast promoter.
 70. The methodof any one of claims 65-69 wherein the bacterial cell or the yeast cellis an E. coli cell or a Saccharomyces cerevisiae cell.
 71. The method ofany one of claims 65-70 wherein the cell-based vaccine formulation isformulated for injection.
 72. The method of any one of claims 65-71further comprising a step of generating a third recombinant nucleic acidthat is a viral expression vector that includes the sequence thatencodes the polytope, and wherein the third recombinant nucleic acidcomprises a third promoter operably linked to the sequence that encodesthe polytope to drive expression of the polytope in a cell of theindividual.
 73. The method of claim 72 wherein the third promoter is aconstitutive promoter or wherein the third promoter is inducible byhypoxia, IFN-gamma, or IL-8.
 74. The method of claim 65 wherein thefirst recombinant nucleic acid is an expression vector.
 75. The methodof claim 65 wherein the trafficking element is selected from the groupconsisting of a CD1b leader sequence, a CD1a tail, a CD1c tail, and aLAMP1-transmembrane sequence.
 76. The method of claim 65 wherein thesecond promoter is a constitutive bacterial or a yeast promoter.
 77. Themethod of claim 65 wherein the the bacterial cell or the yeast cell isan E. coli cell or a Saccharomyces cerevisiae cell.
 78. The method ofclaim 65 wherein the cell-based vaccine formulation is formulated forinjection.
 79. The method of claim 65 further comprising a step ofgenerating a third recombinant nucleic acid that is a viral expressionvector that includes the sequence that encodes the polytope, and whereinthe third recombinant nucleic acid comprises a third promoter operablylinked to the sequence that encodes the polytope to drive expression ofthe polytope in a cell of the individual.
 80. The method of claim 79wherein the third promoter is a constitutive promoter or wherein thethird promoter is inducible by hypoxia, IFN-gamma, or IL-8. 81.(AdV/cell based) A method of preparing first and second treatmentcompositions for an individual having a tumor, comprising: identifying aplurality of expressed neoepitope sequences from omics data of thetumor, wherein the expressed neoepitope sequences have a calculatedbinding affinity of equal or less than 500 nM to at least one of MHC-Iand MHC-II of the individual; generating a first recombinant nucleicacid having a sequence that encodes a polytope, wherein the polytopecomprises the plurality of expressed neoepitope sequences, wherein thefirst recombinant nucleic acid is a viral expression vector; wherein thepolytope further comprises a trafficking element that directs thepolytope to a sub-cellular location selected from the group consistingof a recycling endosome, a sorting endosome, and a lysosome; wherein thefirst recombinant nucleic acid comprises a first promoter operablylinked to the sequence that encodes the polytope to drive expression ofthe polytope in a cell of the individual; forming viral particles fromthe viral expression vector and formulating the viral particles into aviral vaccine formulation to so obtain the first treatment composition;generating a second recombinant nucleic acid having the sequence thatencodes the polytope, wherein the second recombinant nucleic acidcomprises a second promoter operably linked to the sequence that encodesthe polytope to drive expression of the polytope in a non-mammaliancell; transfecting a bacterial cell or a yeast cell with the secondrecombinant nucleic acid and expressing the polytope in the bacterialcell or the yeast cell; and formulating the transfected bacterial cellor the yeast cell into a cell-based vaccine formulation to so obtain thesecond treatment composition.
 82. The method of claim 81 wherein theplurality of expressed neoepitope sequences are identified usingincremental synchronous alignment of omics data from the tumor and omicsdata from a non-tumor sample of the same individual.
 83. The method ofany one of claims 81-82 wherein the trafficking element is selected fromthe group consisting of a CD1b leader sequence, a CD1a tail, a CD1ctail, and a LAMP1-transmembrane sequence.
 84. The method of any one ofclaims 81-83 wherein the first promoter is a constitutive promoter orwherein the first promoter is inducible by hypoxia, IFN-gamma, or IL-8.85. The method of any one of claims 81-84 wherein the viral expressionvector is an adenoviral expression vector, optionally having E1 and E2bgenes deleted.
 86. The method of any one of claims 81-85 wherein thesecond promoter is a constitutive bacterial or a yeast promoter.
 87. Themethod of any one of claims 81-86 wherein the non-mammalian cell or theyeast cell is an E. coli cell or a Saccharomyces cerevisiae cell. 88.The method of any one of claims 81-87 wherein the viral vaccineformulation and the cell-based vaccine formulation are formulated forinjection.
 89. The method of claim 81 wherein the trafficking element isselected from the group consisting of a CD1b leader sequence, a CD1atail, a CD1c tail, and a LAMP1-transmembrane sequence.
 90. The method ofclaim 81 wherein the first promoter is a constitutive promoter orwherein the first promoter is inducible by hypoxia, IFN-gamma, or IL-8.91. The method of claim 81 wherein the viral expression vector is anadenoviral expression vector, optionally having E1 and E2b genesdeleted.
 92. The method of claim 81 wherein the second promoter is aconstitutive bacterial or a yeast promoter.
 93. The method of claim 81wherein the non-mammalian cell or the yeast cell is an E. coli cell or aSaccharomyces cerevisiae cell.
 94. The method of claim 81 wherein theviral vaccine formulation and the cell-based vaccine formulation areformulated for injection.